A developing mammalian fetus receives oxygen and voids carbon dioxide by means of a close juxtaposition of its own blood vessels with those of its mother’s circulatory system in the placenta. The fetus’s heart pumps blood through the umbilical cord to the placenta, where that blood picks up O2 that its mother’s blood has brought there from her lungs. The now- oxygenated fetal blood returns to the fetus through the umbilical cord and is circulated to all the parts of the body of the fetus. By 10 weeks after conception, a human fetus already has hemoglobin-rich blood. At that age, the amount of hemoglobin per unit of volume in the fetus’s blood has reached 50% of the adult concentration and is increasing rapidly, so that it will be about 80% of the adult value at 20 weeks of age.

In the placenta, O2 must cross from the mother’s blood to the fetus’s blood by diffusion through tissues separating the two circulatory systems. The detailed way in which this oc- curs remains a topic of active research. The basic options for the mode of gas transfer are countercurrent gas exchange, cross-current gas exchange, and cocurrent (concurrent) gas exchange—the same options we discussed in Chapter 23 (pages 602–603) for the transfer of O2 between fluid streams. The mode of gas transfer in the human placenta remains un- certain for two reasons. First, experiments cannot be done on human fetuses. Second, other

A human fetus obtains oxy- gen (O2) by pumping blood along its umbilical cord to
the placenta, where its blood picks up O2 from its mother’s blood Hemoglobin plays a major role in the acquisition and transport of O2 by the fetus. In the mother,

O2 taken up in her lungs combines with hemoglobin in her red blood cells and is carried by blood flow
to the placenta in that form. In the placenta, the fetus’s hemoglobin combines with O2 that is released from the mother’s hemoglobin.The O2 combined with fetal hemoglobin is then carried, by the circulation of the fetus’s blood, from the placenta into the body of the fetus, where the O2 is used.

636 Chapter 24

species of placental mammals exhibit such wide diversity in the morphology and physiology of their placentas that researchers are not certain which animal model would best reveal how the human placenta works. Enough is known about placental physiology in several mammalian species to make clear, however, that—contrary to expectation—substantial impediments to O2 transfer from ma- ternal to fetal blood often exist in mammalian placentas.1 Placentas are emphatically not like lungs, in which high rates of air and blood flow and minutely thin intervening membranes result readily in dramatic blood oxygenation.

An important reason a human fetus can in fact obtain enough O2 from its placenta is that the fetus produces a different molecular form of hemoglobin from the one its mother produces. This is also true in many other species of placental mammals that have been studied. For reasons we discuss principally later in this chapter, fetal hemoglobin has a higher affinity for O2 than adult hemo- globin does. This greater affinity has two important, interrelated consequences. First, the difference in affinity between the maternal and fetal hemoglobins means that O2 has a chemical tendency to leave the lower-affinity hemoglobin of the mother to bind with the higher-affinity hemoglobin of the fetus. Second, the high absolute affinity of fetal hemoglobin means that it can become well oxygenated even if the O2 partial pressure in the fetal blood remains relatively low, as it typically does.

The hemoglobins are one of several types of respiratory pig- ments or oxygen-transport pigments that animals have evolved. The defining property of the respiratory pigments is that they un- dergo reversible combination with molecular oxygen (O2). Thus they can pick up O2 in one place, such as the lungs of an adult or the placenta of a fetus, and release the O2 in another place, such as the systemic tissues2 of the adult or fetus. All the types of respiratory pigments are metalloproteins: proteins that contain metal atoms, exemplified by the iron in hemoglobin. In addition, all are strongly colored at least some of the time, explaining why they are called pigments.

The most straightforward function of the respiratory pigments is to increase the amount of O2 that can be carried by a unit of volume of blood. Although O2 dissolves in the blood plasma3 just as it dis- solves in any aqueous solution (see Chapter 22), the solubility of O2 in aqueous solutions is relatively low, meaning that the amount of O2 that can be carried in dissolved form per unit of volume is not high. When a respiratory pigment is present in the blood, however, the blood can carry O2 in two ways: in chemical combination with the pigment as well as in simple solution. Therefore a respira- tory pigment increases the oxygen-carrying capacity of blood, meaning the total amount of O2 that can be carried by each unit of volume. In some cases, the increase is very large. For example, when the blood of an adult person leaves the lungs, it contains

1 The placental O2 partial pressure is strikingly low during the first trimester of human intrauterine development, for example. To explain this unexpected state, one hypothesis is that the low partial pressure helps limit formation of reactive oxygen species (see Box 8.1), which might be particularly damaging to the early developmental stages.

2 The systemic tissues are all tissues other than the tissues of the breathing organs.

3 The plasma of the blood is the aqueous solution in which the cells are suspended. Operationally, plasma is obtained by removing all cells from blood (e.g., by centrifugation).

almost 200 mL of O2 per liter of blood in chemical combination with hemoglobin and about 4 mL of O2 per liter in solution. Thus the blood’s concentration of O2 is increased about 50-fold by the presence of hemoglobin. This means, among other things, that the heart can work far less intensely; roughly calculated, the circulation of 1 L of actual human blood delivers the same amount of O2 as would the circulation of 50 L of blood without hemoglobin.

Multiple molecular forms of hemoglobin occur, as already exem- plified by the contrast between fetal and maternal hemoglobins. Not only may one species have multiple molecular forms, but different species have different forms. Thus the word hemoglobin refers to a family of many compounds, not just a single compound. To empha- size this fact, we refer to these compounds as hemoglobins (plural) rather than just hemoglobin (singular). All the hemoglobins—plus a great diversity of other globin proteins—are coded by genes of a single ancient gene family. Natural selection and other processes have modified the genes in this gene family over evolutionary time, giving rise to the great diversity of hemoglobins and other globin proteins in modern organisms.

Hemoglobins have several functions; that is, their functions are not limited just to increasing the blood’s oxygen-carrying capacity. Blood hemoglobins, for example, play important roles as buffers and participate in blood CO2 transport as well as O2 transport. Moreover, specialized hemoglobins are found within muscle cells or nerve cells (neurons), where they often facilitate diffusion of O2 into the cells and potentially serve as intracellular storage depots for O2. Fast-breaking research indicates also that hemoglobins within some muscle cells serve in intricate ways both to synthesize and break down intracellular nitric oxide (NO), a compound that potently controls mitochondrial respiration (mitochondrial O2 consumption and ATP production) in the muscle cells. This chapter emphasizes the role of hemoglobins in blood O2 transport but touches on the other functions as well.

One could aptly say that a revolution is currently underway in the study of the respiratory pigments. The driving forces in this revolution are molecular sequencing, genomics, applications of advanced chemical analysis, and phylogenetic reconstruction. For instance, because of the availability of relatively cheap molecular sequencing tools, it is becoming routine—as it has not been before—to know the entire amino acid sequences of respiratory- pigment molecules that are being compared. Genomics facilitates the widespread search for respiratory-pigment molecules and has led to the discovery of new ones.

A final introductory point worth noting is that when hemoglobins or other respiratory pigments combine with O2, they are said to be oxygenated, and when they release O2, they are deoxygenated. They are not said to be oxidized and reduced. The reason for these distinctions is that the process by which a respiratory pigment combines with O2 is not chemically equivalent to oxidation. During the oxygenation of a hemoglobin molecule, for example, although electrons are partially transferred from iron atoms in the hemoglobin molecule to the O2, the transfer is not complete, as it would be in full-fledged oxidation. In fact, if a hemoglobin molecule accidentally becomes truly oxidized (so that its iron atoms are converted from their ordinary ferrous state to the ferric state), the molecule (now called methemoglobin) loses its ability to combine with O2! The prefixes oxy- and deoxy- are used to specify the oxygenated and deoxygenated states of respiratory-pigment molecules. Hemoglobin,

Transport of Oxygen and Carbon Dioxide in Body Fluids 637

for example, is called oxyhemoglobin when it is combined with O2 (oxygenated) and deoxyhemoglobin when it is not combined with O2 (deoxygenated). Respiratory pigments change color when they are oxygenated and deoxygenated, and measures of these color changes can be used to monitor the oxygenation and deoxygenation of blood (BOx 24.1).

The Chemical Properties and Distributions of the Respiratory Pigments

Four chemical categories of respiratory pigments are recognized: hemoglobins, hemocyanins, hemerythrins, and chlorocruorins. The prefix hemo- is from the Greek for “blood,” explaining its use in the names of three of the pigment categories. Like the hemo- globins, the other categories are groups of related compounds, not single chemical structures.

Many of the important chemical properties of the respira- tory pigments resemble the properties of the enzyme proteins we studied in Chapter 2. The parallels are so great, in fact, that biochemists have occasionally dubbed the respiratory pigments “honorary enzymes.” The point of mentioning these parallels is not to suggest that respiratory pigments are enzymes; in terms oftheirprincipalfunctions,theyarenot.Thepoint,instead,isto highlight that, based on your knowledge of enzyme proteins, you will find that you already know a great deal about the molecular features of respiratory pigments.

When a hemoglobin molecule, for example, combines with O2, it does so at defined binding sites, resembling the way in which

enzymes combine with their substrates at defined binding sites. Moreover, the combination of the O2-binding sites with O2 is highly specific and occurs by noncovalent, weak bonding (see Box 2.1), just as enzyme–substrate binding is specific and noncovalent. Accordingly, O2 is a ligand of hemoglobin, based on the definition of “ligand” we developed in Chapter 2. When a hemoglobin molecule combines with O2, it undergoes a change in its molecular conformation (shape) that is analogous to the conformational change an enzyme molecule undergoes when it combines with its substrate; the ability of a hemoglobin molecule to flex in this way is an essential attribute of its function, just as molecular flexibility is critical for enzyme function. One of a hemoglobin molecule’s most important proper- ties is its affinity for O2, meaning the ease with which it binds with the O2 molecules it encounters; thus a hemoglobin molecule (like an enzyme) is characterized in part by how readily it binds with its primary ligand.

A hemoglobin molecule also has specific sites at which it combines with ligands other than O2. Using the same terminology we used in Chapter 2 in connection with enzymes, such ligands (e.g., H+ and CO2) are allosteric ligands or allosteric modulators, because when they bind with their specific sites on a hemoglo-

bin molecule, they affect the ability of the hemoglobin to bind

Hill Animal Physiology 4E
with its primary ligand, O . Allosteric ligands, for example, can

Sinaue2r Associates
potently affect a hemoglobin molecule’s affinity for O . Within a

Morales Studio hemoglobinmoleculeF,ijgusretBasoxin24a.0n1en12z-y1m1-e15molecule,allosteric

ligands exert their effects at a distance; that is, the binding sites for allosteric ligands on a hemoglobin molecule are separate from the O2-binding sites, and when allosteric ligands affect O2 binding, they do so by modifying the conformation and flexibility of the molecule as a whole.

2

638 Chapter 24

Hemogloblin molecules are usually multisubunit proteins; that is, each whole molecule consists of two or more proteins bonded together by noncovalent bonds. Multisubunit hemoglobins have an O2-binding site on each subunit and thus have multiple O2-binding sites. In com- mon with multisubunit enzymes that exhibit cooperativity among substrate-binding sites, multisubunit hemoglobin molecules exhibit cooperativity among their O2-binding sites, meaning that binding of O2 to any one site on a molecule affects how readily the other sites bind O2.4 These interactions among O2-binding sites themselves occur at a distance; the various O2-binding sites on a multisubunit hemoglobin are separate and distinct, and they influence each other by effects that are relayed through the structure of the whole multisubunit molecule, rather than by direct site-to-site effects.

The points we have made using hemoglobin as an example apply to the other categories of respiratory pigments as well. Thus, in the study of all respiratory pigments, it is helpful to keep these points in mind.

Despite sharing many key properties with enzymes, the respi- ratory pigments differ from enzymes in a major way: They do not modify their primary ligand. After they combine with O2, they later release the same molecule, O2.

Hemoglobins contain heme and are the most widespread respiratory pigments

The chemical structures of all hemoglobin molecules share two features. First, all hemoglobins contain heme (FiguRE 24.1A), which is a particular metalloporphyrin containing iron in the ferrous state (ferrous protoporphyrin IX). Second, the heme is noncovalently bonded to a protein known as a globin (FiguRE

4 In the terminology developed in Chapter 2, this is homotropic cooperativity. See page 49 for more on cooperativity within multisubunit proteins.

24.1B). The combination of heme with globin accounts for the name hemoglobin. Oxygen binds at the heme site at a ratio of one O2 molecule per heme. In all hemoglobin molecules, the heme is identical. The multiple molecular forms of hemoglobin differ in their protein (globin) structures (and in the numbers of unit molecules of hemoglobin that are linked together).

Biochemical studies reveal that small changes in the protein structure of a hemoglobin molecule can cause highly significant alterations in the functional properties of the molecule. There are, to illustrate, more than 100 known mutant forms of human hemoglobin. Each human globin protein consists of more than 140 amino acids, and most of the mutant forms differ from the normal form in just one of those amino acids. Nonetheless, many mutant forms differ markedly from the normal form in their functional properties; they may differ in their affinity for O2 or in other key properties, such as their solubility or structural stability.

The blood hemoglobins of vertebrates are almost always four- unit (tetrameric) molecules (FiguRE 24.1C) that can bind a total of four O2 molecules. The molecular weight of each unit molecule is typically about 16,000–17,000 daltons (Da). Thus the four-unit blood hemoglobins have molecular weights of approximately 64,000–68,000 Da. Two types of globins, termed α and β, are found in adult blood hemoglobins. Most biochemists agree that the ancestral genes for the two types originated by gene duplica- tion about 500 million years ago. Each molecule of adult blood hemoglobin consists of two α units and two β units. The human α-globin contains 141 amino acids, whereas the human β-globin contains 146 amino acids. Although other vertebrate species are also described as having α- and β-globins, the particular chemical structures of those globins vary from species to species. Relatively huge hemoglobin molecules are found in the blood of some inver- tebrates, as we will soon see.

(A) Heme

HC H3C C C

H2C C C

H CH3 C

CC

CH2

CH
C C

(B) Whale myoglobin: An example of a single heme–globin complex

(C) Mammalian adult blood hemoglobin: A tetramer consisting of four heme–globin subunits

Heme

The iron and nitrogen atoms in heme form a planar structure.

β-Globin

β-Globin

C

C

N
N Fe N N

CH3
C C C CH2

CH2 HC C C CH COOH C C

CH2 CH3 H2C COOH

Globin

O binds here. H2

FiguRE 24.1 The chemical structure of hemoglobin
structure of heme: Ferrous iron forms a complex with protoporphyrin. The positions assigned to double and single bonds in the porphyrin ring are arbitrary because resonance occurs. (B) A single heme–globin complex.The specific molecule shown is myoglobin (muscle hemo- globin) taken from the muscle of a whale.The structure of the globin protein includes eight segments in which the amino acid backbone of

(A) The

α-Globin
the protein (seen spiraling inside the cylindrical outline) forms a helix.

The outer, cylindrical part of the drawing shows the major contours of the globin structure. (C) A tetrameric hemoglobin molecule of the sort found in mammalian red blood cells. In adults, each tetramer consists of two α-globins, two β-globins, and a total of four heme groups. (B and C after Dickerson and Geis 1983.)

α-Globin

50 40 30 20 10

0

6 12 18 24 30 36 Time (weeks) between

conception and birth

6 12 18 24 30 36 42 48 Postnatal age (weeks)

types of globins synthesized for incorporation into blood hemoglobins Each blood-hemoglobin molecule consists of four globin units plus four O2-binding heme groups (see Figure 24.1C). In early embryos, the principal globins syn- thesized are α-globin, ε-globin (epsilon-globin), and ζ-globin (zeta-globin); embryonic hemoglobins are made up princi- pally of these globins (e.g., one common form consists of two α-globin and two ε-globin units). By about 8 weeks after con- ception, α-globin and γ-globin (gamma-globin) are the princi- pal globins synthesized, and most hemoglobin molecules are fetal hemoglobin molecules consisting of two α-globin and two γ-globin units. Following birth, synthesis of γ-globin ebbs, whereas that of β-globin increases, so that by 20 weeks of postnatal age, the blood hemoglobin is predominantly adult hemoglobin consisting of α- and β-globins.The dashed part of the β-globin curve is postulated rather than empirical. (After Wood 1976.)

Embryonic globin synthesis

Fetal globin synthesis

Transport of Oxygen and Carbon Dioxide in Body Fluids 639 FiguRE 24.2 Human developmental changes in the

Animals sometimes have hemoglobins inside muscle cells or inside the cells of other tissues besides blood. Such hemoglobins typically differ from blood hemoglobins in their chemical structure. The muscle hemoglobins, termed myoglobins (myo-, “muscle”), of vertebrates provide apt examples. Located in the cytoplasm of muscle fibers (muscle cells), they tend to be especially abundant in cardiac muscle fibers and in the slow oxidative (SO) class of skeletal muscle fibers (see page 202). When present at high concentrations, they impart a reddish color to the tissue; “red” muscles are red because of myoglobins. Unlike blood hemoglobins, vertebrate myoglobins appear always to be single-unit (monomeric) molecules (see Figure 24.1B). They also have distinctive globins. In adult humans, for example, the globin of myoglobin is of different structure than the α- or β-globins.

In addition to varying spatially—from tissue to tissue—within an animal, the chemical nature of hemoglobin often also changes temporally over the life cycle. For example, as already mentioned, the blood hemoglobin of fetal mammals is often different from that of the adults of their species. In humans, fetuses synthesize α-globin (as adults do) and γ-globin (gamma-globin), which dif- fers from the β-globin synthesized by adults (FiguRE 24.2); each fetal blood-hemoglobin molecule consists of two α-globin and two γ-globin subunits. Earlier in development, as Figure 24.2 shows, still different globins are expressed.

Although the adult blood hemoglobin of many animals (e.g.,

humans and most other mammals) is of essentially uniform

composition, in many species of poikilothermic vertebrates and

invertebrates, the blood of adults normally consists of mixes of two,

three, or even ten or more chemically different forms of hemoglobin.

A relatively simple example is provided by the blood hemoglobin

of the sucker fish Catostomus clarkii, which consists of about 80%

protists and plants. They are the only respiratory pigments found in vertebrates, and with a few interesting exceptions (see Chap- ter 3), all vertebrates have hemoglobin in their blood. The blood hemoglobins of vertebrates are always contained in specialized cells, the red blood cells (erythrocytes), discussed in BOx 24.2.

Among the invertebrates, the distribution of hemoglobins is not only wide but sporadic. Hemoglobins may occur within certain subgroups of a phylum but not others, and even within certain spe- cies but not other closely related species. Sometimes, among all the members of a large assemblage of related species, only an isolated few possess hemoglobins. The evolution of the wide but sporadic distribution of hemoglobins certainly provokes curiosity. According to the prevailing view at present, the hemoglobin gene family origi- nated even before animals did, and therefore genes of the family are potentially present in all evolutionary lines of animals. The genes are sometimes fully functional and expressed in modern animals, and sometimes not—accounting for the hemoglobin distribution observed.

The circulating hemoglobins of invertebrates may be found in blood, or they may occur in other moving fluids, such as coelomic fluids. Sometimes, as in vertebrates, these hemoglobins are contained within cells and thus categorized as intracellular. The intracellular hemoglobins of invertebrates are always of relatively low molecular weight (~14,000–70,000 Da); structurally, they are generally one-, two-, or four-unit molecules. By contrast, the blood hemoglobins of some invertebrates are dissolved in the blood plasma and thus categorized as extracellular. Earthworms (Lumbricus), for example, have hemoglobin dissolved in their blood plasma, which when held to the light is wine red and clear—quite unlike vertebrate bloods, which are opaque because of their high concentrations of red blood cells. The extracellular, dissolved hemoglobins of invertebrates are—almostalways—relativelyhuge,multiunitmolecules,having molecular weights of 0.2–12 million Da. There are 144 O2-binding sites in each molecule of earthworm (Lumbricus) hemoglobin!

The concentration of blood hemoglobin in some invertebrates changes so dramatically from one environment to another that the animals change color. Water fleas (Daphnia), for example, have little hemoglobin and are pale when they have been living in O2-rich waters. However, if they are placed in O2-poor waters, they increase their levels of hemoglobin within days and become bright red (see Figure 24.20).

ofHoillneAmnimajaolrPthyypsieologfyh4eEmoglobinand20%ofanother.Whenmul- Sinauer Associates

tiple chemical forms of hemoglobin occur in a species, the forms

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sometimes differ substantially in their O2-binding characteristics.

Figure 24.01 12-10-15

Possession of multiple blood hemoglobins may thus permit a species to maintain adequate O2 transport over a broader range of condi- tions than would be possible with only a single hemoglobin type.

THE DiSTRiBuTiOn OF HEmOglOBinS Hemoglobins are the most widely distributed of the respiratory pigments, being found in at least nine phyla of animals (FiguRE 24.3) and even in some

Birth

Percentage of total globin synthesis

640 Chapter 24

FiguRE 24.3 The distribution of the two major respiratory pig- ments—hemoglobins and hemocyanins—in animals A red square indicates that hemoglobins occur in solid tissues such as muscle or nerve tissues. Red circles indicate that hemoglobins occur in circulat- ing body fluids. A single small red circle indicates the presence in circulat- ing body fluids of hemoglobins consisting of one unit molecule of heme plus globin: hemoglobin monomers. A pair or foursome of small red circles symbolizes hemoglobin dimers or tetramers, respectively. A large red circle indicates polymeric hemoglobins of high molecular weight, consisting of many joined unit molecules.The polymeric hemoglobins are always ex- tracellular—dissolved in the circulating fluid. The monomeric, dimeric, and tetrameric forms of hemoglobin, with few exceptions, are intracellular— contained within circulating cells such as erythrocytes.A large blue circle indicates polymeric hemocyanins of high molecular weight dissolved

in the blood; this is the only circumstance in which hemocyanins occur.
In each group labeled here as having hemoglobin or hemocyanin, not all species in the group have it; nor does each species with hemoglobin necessarily have all the chemical forms of hemoglobin shown. In verte- brates, for example, although most have blood hemoglobin, icefish do not (see Chapter 3); and the monomeric and dimeric forms of blood hemo- globin occur only in cyclostome fish, with tetrameric forms being found in all other vertebrates that have blood hemoglobin. Similarly, whereas some arthropods and molluscs have hemoglobins as symbolized here, the ma- jority lack them.This summary is not exhaustive,and it assumes echiurids are annelids. (Hemoglobin data from Terwilliger 1980; cladogram after Sadava et al. 2008.)

Copper-based hemocyanins occur in many arthropods and molluscs

Hemocyanins are found in just two phyla—the arthropods and the molluscs (see Figure 24.3)—but clearly rank as the second most common class of respiratory pigments. In turning to the hemocya- nins, we encounter a minor problem that they share with the chlo- rocruorins and hemerythrins: The names given to these compounds provide no clue to their chemical structures. Hemocyanins do not contain heme, iron, or porphyrin structures. The metal they contain is copper, bound directly to the protein. The arthropod and mol- lusc hemocyanins exhibit consistent structural differences and are clearly of separate evolutionary origin. Thus they are distinguished as arthropod hemocyanins and mollusc hemocyanins. Each O2-binding site of a hemocyanin contains two copper atoms; thus the binding ratio is one O2 molecule per two Cu. In both phyla, hemocyanins are invariably found dissolved in the blood plasma, not in cells, and are typically large molecules (4–9 million Da in molluscs, 0.5–3 mil- lion Da in arthropods) that have numerous O2-binding sites. The number of binding sites per molecule is as high as 160 in some cases. Although hemocyanins are colorless when deoxygenated, they turn bright blue when oxygenated. Species that have high concentrations of hemocyanins are dramatically blue-blooded!

The molluscs that possess hemocyanins include the squids and octopuses (cephalopods), many chitons and gastropods (snails and slugs), and a relatively small subset of bivalves. Hemocyanins are not present in most bivalve molluscs (clams, scallops, and the like); indeed, bivalves usually lack circulating respiratory pigments of any kind. Groups of arthropods in which hemocyanins are important include the decapod crustaceans (crabs, lobsters, shrimps, and crayfish), the horseshoe crabs, and the spiders and scorpions. Even some (relatively primitive) insects have recently been discovered to have hemocyanins. Hemocyanins are never found within muscle or other solid tissues. Certain molluscs that have blood hemocyanins have hemoglobins in their muscles, neurons, or gills.

Deuterostomes

KEY

Sponges Ctenophores Cnidarians Chaetognaths Ectoprocts Flatworms Rotifers Nemerteans Brachiopods Phoronids Annelids
Molluscs Priapulids Kinorhrynchs Loriciferans Nematomorphs Nematodes Onychophorans Tardigrades Arthropods Hemichordates Echinoderms Urochordates Cephalochordates Vertebrates

Hemoglobins—usually single-unit molecules—are found widely in solid tissues of invertebrates, not only in muscles but also in certain other tissues. Both muscle and nerve hemoglobins occur, for example, in a wide variety of molluscs and annelids; the nerve hemoglobins may be present in neurons per se or in support cells (glial cells), but either way, they sometimes impart a striking pinkish or red color to the ganglia or nerves. Although insects usually lack circulating respiratory pigments, large numbers of insect species hHailvl e AhneimaolgPlhoybsionlosgiyn4tEhe fat body or parts of the tracheal system.

Such hemoglobins in some backswimmer bugs, for example, store

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O2 for release to the tracheae during diving.

Figure 24.03 12-10-15

Ecdysozoans

Protostomes Lophotrochozoans

Hemoglobin in muscle, neurons, neuron-support cells (glial cells), or other solid tissue

Hemoglobin in blood or other circulating fluid: Monomers

Dimers Usually intracellular Tetramers
Polymers – Extracellular

Hemocyanin in blood
Polymers – Extracellular

Figure Box 24.02 12-11-15

Chlorocruorins resemble hemoglobins and occur in certain annelids

Chlorocruorins, also sometimes called green hemoglobins, occur in just four families of marine annelid worms, including the fan worms and feather-duster worms that are so popular with aquar- ists. Chlorocruorins are always found extracellularly, dissolved in the blood plasma. They have close chemical similarities to the extracellular hemoglobins found dissolved in the blood plasma of many other annelids. Like the extracellular hemoglobins, they are large molecules, with molecular weights of close to 3 million Da, composed of unit molecules consisting of iron-porphyrin groups conjugated with protein. They bind one O2 per iron-porphyrin group. The chlorocruorins differ from hemoglobins in the type of iron porphyrin they contain.5 This difference gives the chloro- cruorins a distinctive and dramatic color. In dilute solution, they are greenish. In more concentrated solution, they are deep red by transmitted light but greenish by reflected light.

iron-based hemerythrins do not contain heme and occur in three or four phyla

Hemerythrins have a distribution that is puzzling because it is both limited and far-flung, encompassing three or four phyla. Cir-

5 The porphyrin differs from heme in that one of the vinyl chains (—CH=CH2) on the periphery of the protoporphyrin ring in heme (see Figure 24.1A) is replaced with a formyl group (—CHO).

culating hemerythrins occur in a single family of marine annelid worms (the magelonids), in the sipunculid worms (which have been a separate phylum but might be annelids), in many brachiopods (lamp shells), and in some species of the small phylum Priapulida. Despite their name, hemerythrins do not contain heme. They do contain iron (ferrous when deoxygenated), bound directly to the protein. Each O2-binding site contains two iron atoms, and there is one such site per 13,000–14,000 Da of molecular weight. In some instances (including, for example, some annelids that lack circulating hemerythrin), single-unit hemerythrins, known as myohemerythrins, occur within muscle cells. Better known are the circulating hemerythrins, which are always located intracellularly, in blood or coelomic cells, and typically have molecular weights of 40,000–110,000 Da; many are octomers, having eight O2-binding sites per molecule. Hemerythrins are colorless when deoxygenated but turn reddish violet when oxygenated.

Transport of Oxygen and Carbon Dioxide in Body Fluids 641

642 Chapter 24

(A) Percentage of heme groups oxygenated as a function of O2 partial pressure

The O2-Binding Characteristics of Respiratory Pigments

A key tool for understanding the function of a respiratory pigment is the oxygen equilibrium curve. In a body fluid containing a respiratory pigment, there is a large population of O2-binding sites. Human blood, for example, contains about 5.4 × 1020 heme groups per 100 mL. The combination of O2 with each individual O2-binding site is stoichiometric: One and only one O2 molecule can bind, for example, with each heme group of a hemoglobin or with each pair of copper atoms in a hemocyanin. However, in blood, where there are great numbers of O2-binding sites, all sites do not simultaneously bind with O2 or release O2. Instead, the fraction of the O2-binding sites that are oxygenated varies in a graded manner with the O2 partial pressure. The oxygen equilibrium curve, also sometimes called the oxygen dissociation curve, shows the functional relation between the percentage of binding sites that are oxygenated and the O2 partial pressure (FiguRE 24.4A).

The respiratory pigment in the blood of an animal is said to be saturated if the O2 partial pressure is high enough for all O2-binding sites to be oxygenated. Accordingly, the percentage of binding sites that are oxygenated is often termed the percent saturation. The blood’s oxygen-carrying capacity, an important property mentioned earlier, is the amount of O2 carried per unit of volume at saturation. Ordinary human blood, for example, has an oxygen-carrying capacity of about 20 mL O2 per 100 mL of blood

20 15 10

5

0 0

20 40 60

This plateau (relatively flat part) in the oxygen equilibrium curve means that a wide range of blood O2 partial pressures is sufficient to cause virtually full hemoglobin saturation.

80 100 120 mm Hg

(B)

0 4 8 12 16kPa Partial pressure of O2 in blood

Blood O2 concentration as a function of O2 partial pressure

100 80 60 40 20

00 20 40 60

80 100

120 mm Hg

This oxygen equilibrium curve shows the total amount of O2 per unit of blood volume, including both hemoglobin-bound and dissolved O2.

0 4 8 12 16kPa Partial pressure of O2 in blood

FiguRE 24.4 A typical oxygen equilibrium curve for human arterial blood presented in two different ways (A) The per- centage of heme groups oxygenated as a function of the O2 partial pressure. (B) The total blood O2 concentration—including both hemoglobin-bound and dissolved O2—as a function of the O2 partial pressure; the portion of the total O2 present as dissolved O2 is plotted at the bottom. Normal arterial values of CO2 partial pressure, pH, and temperature are assumed. In humans, as in other animals, significant individual variation occurs. Partial pressure is shown in two systems of units, the SI system (kPa) and a traditional system (mm Hg)—as will often be the case in this chapter. (After Roughton 1964; B assumes an O2 concentration of 20 vol % at 16 kPa.)

and actually has that concentration of O2 when saturated.6 The volume of gas carried per 100 volumes of blood is often termed the volumes percent (vol %) of gas. In that system of units, the oxygen-carrying capacity of ordinary human blood is 20 vol %.

6 Gas volumes are always expressed at standard conditions of temperature

Hill Animal Physiology 4E
and pressure (see Appendix C) unless otherwise stated. Such volumes are

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proportional to molar quantities, as discussed in Chapter 22. Morales Studio

Figure 24.04 12-10-15

Oxygen equilibrium curve (total O2)

Dissolved O2

mL of O2 per 100 mL of blood (vol %) Percentage of heme groups oxygenated

The oxygen equilibrium curve can be presented in two ways. Figure 24.4A—showing the percentage of oxygenated binding sites (the percent saturation) as a function of O2 partial pressure—exemplifies one of these. The alternative presentation, seen in FiguRE 24.4B, shows the blood O2 concentration as a function of the O2 partial pres- sure. To calculate this alternative form of the curve from the first form, one needs merely to convert the percentage of oxygenated binding sites at each partial pressure into the corresponding blood O2 concentration. For most purposes, this conversion can be carried out by use of the oxygen-carrying capacity: The O2 concentration at each partial pressure is the oxygen-carrying capacity multiplied by the percentage of oxygenated binding sites.7

Because O2 dissolves in the blood plasma, blood in fact contains O2 in two forms: dissolved and bound to the respiratory pigment. The amount of dissolved O2 per unit of blood volume simply fol- lows the principles of gas solution (see Equation 22.3). Therefore it is proportional to the O2 partial pressure, producing a straight-line relation as seen at the bottom in Figure 24.4B. In humans and most other vertebrates, dissolved O2 accounts for just a small fraction of all O2 in the blood (see Figure 24.4B).

Later in this chapter, we will see that the O2-binding properties of respiratory pigments are often affected by temperature, pH, and other properties of the blood chemical environment. We will also discuss the reason for the sigmoid shape of the oxygen equilibrium curve (see Figure 24.4). Before we consider those factors, however, it is important to understand the basic principles of respiratory-pigment function in living animals and to appreciate the interpretive value of oxygen equilibrium curves. To these ends, and recognizing that refinements will later be needed, let’s look at the fundamentals of O2 transport by our own blood.

partial pressures; provided the blood O2 partial pressure is high enough to be in the plateau region, hemoglobin will be almost saturated with O2 regardless of the partial pressure. The alveolar O2 partial pressure could even vary a bit, and still, because of the plateau—a property of the hemoglobin—the blood leaving the lungs would remain almost entirely saturated. The close “matching” of the saturation partial pressure of hemoglobin and the alveolar partial pressure represents a striking evolutionary coadaptation: The hemoglobin molecule has evolved O2-binding properties that suit it to oxygenate well at the O2 partial pressures maintained in the lungs by the breathing system.

After leaving the lungs, blood flows to the left side of the heart and is pumped to the systemic tissues. To understand the events in the systemic tissues, it is crucial to recall that in the mitochondria, O2 is continually being combined with electrons and protons to form H2O. By this process, O2 molecules are removed from solution, and the O2 partial pressure in and around the mitochondria is lowered. Blood arriving in capillaries of systemic tissues from the lungs has a high O2 partial pressure; O2 thus diffuses from the blood to the mitochondria (see Figure 22.7). During this diffusion, dissolved O2 leaves the blood, and the O2 partial pressure of the blood falls. As this occurs, hemoglobin releases (unloads) O2, thereby making hemoglobin-bound O2 available to diffuse to the mitochondria. The oxygen equilibrium curve (see Figure 24.4) is a key to understand- ing the unloading of O2 from hemoglobin: As the blood O2 partial pressure falls, the amount of O2 released from hemoglobin at each O2 partial pressure is dictated by the curve.

Knowing that hemoglobin leaves the lungs in a virtually saturated condition, we can calculate its yield of O2 to the systemic tissues by obtaining a measure of its degree of saturation after it has passed through the systemic tissues. The simplest way to obtain this measure is to determine the degree of saturation in blood drawn from the great veins leading back to the heart; such blood is termed mixed venous blood because it represents a mixture of the venous blood coming from all parts of the body. In people at rest, the O2 partial pressure of mixed venous blood is about 5.3 kPa (40 mm Hg). From the oxygen equilibrium curve (see Figure 24.4B), we can see that blood at this partial pressure contains about 15 mL of O2 /100 mL. Recalling that arterial blood contains about 20 mL of O2/100 mL, we see that the O2 content of the blood falls by about 5 mL of O2/100 mL when the blood circulates through the systemic tissues in humans at rest. In other words, as shown by the “Rest” arrow in FiguRE 24.5, about 5 mL of O2 is released from each 100 mL of blood. The release of O2 from the blood is often expressed as the blood oxygen utilization coefficient, defined to be the percentage of arterial O2 that is released to the systemic tissues. In people at rest, recognizing that arterial blood contains about 20 mL of O2/100 mL and that about 5 mL of O2/100 mL is released to the tissues, the oxygen utilization coefficient is about 25%. That is, only one-fourth of the O2 brought to the systemic tissues in the arterial blood is actually used at rest.

THE SigniFiCAnCE OF mixED vEnOuS O PARTiAl PRES- 2

SuRE The O2 partial pressure of mixed venous blood represents an average of the O2 partial pressures of blood leaving the various systemic tissues. It thus allows us to gauge the blood’s overall drop

Human O
instructive case study

2

transport provides an

To understand the uptake of O2 by the blood in a person’s lungs, it is important to recall from Chapter 23 that breathing maintains the O2 partial pressure in the alveolar gases of our lungs at about 13.3 kPa (100 mm Hg). Blood arriving at the alveoli has a lower O2 partial pressure. Thus O2 diffuses into the blood from the alveolar gas, raising the blood O2 partial pressure as the blood passes through the lungs. As the blood O2 partial pressure rises, at each partial pressure the hemoglobin in the blood takes up the amount of O2 that is dictated by its oxygen equilibrium curve (see Figure 24.4).8

If, in the lungs, the O2 partial pressure of the blood were to rise to the alveolar partial pressure, 13.3 kPa (100 mm Hg), we can see from Figure 24.4 that the hemoglobin in the blood would become virtually saturated with O2. In fact, mixed blood leaving the lungs is at a somewhat lower O2 partial pressure: 12.0–12.7 kPa (90–95 mm Hg) in a person at rest. This lower partial pressure hardly affects the blood O2 content, however, because as shown by the oxygen equilibrium curve, there is a plateau in the relation between the blood O2 concentration and the O2 partial pressure at these high

7 For exacting work, the dissolved O2, discussed in the next paragraph, has to be calculated separately from the pigment-bound O2 and the two amounts added.

8 Hemoglobin also plays an important role in speeding the uptake of O2 by the blood, as discussed in Chapter 22 (see page 592).

Transport of Oxygen and Carbon Dioxide in Body Fluids 643

644

Chapter 24

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DElivERy OF O2 DuRing ExERCiSE As discussed in Chapter 23, controls on breathing tend to keep the alveolar O2 partial pressure stable, near 13.3 kPa (100 mm Hg), as people exercise more and more intensely. During vigorous exercise, however, the blood O2 partial pressure reached in the lungs tends to decline, compared with the resting blood partial pressure; this decline occurs in part because blood passes through the pulmonary circulation faster as the intensity of exercise increases, thus decreasing the time available for equilibration between the alveolar gas and blood. The relative flatness of the oxygen equilibrium curve at high O2 partial pressures again comes to the rescue (see Figure 24.4). Even if the blood passing through the lungs reaches a partial pressure of only 11 kPa (80 mm Hg)—which is often the case during intense exercise—the O2 content of the arterial blood is hardly affected. For simplicity, we treat the O2 content of the arterial blood as a constant as we discuss exercise in more detail.

The modest utilization of blood O2 at rest leaves a large mar- gin to increase utilization during exercise. As we have seen, only about 25% of the O2 carried by the systemic arterial blood is used when people are at rest. The remaining amount, the amount of O2 in mixed venous blood, is called the venous reserve. During exercise, more O2 is withdrawn from each unit of blood volume as the blood passes through the systemic tissues, and the venous reserve becomes smaller.

AhighlysignificantattributeofbloodO transportduringrest 2

is that the resting mixed venous O2 partial pressure, averaging 5.3 kPa (40 mm Hg), is low enough to be below the plateau displayed by the oxygen equilibrium curve at high partial pressures (see the green-shaded part of the curve in Figure 24.5). During exercise, therefore, when the venous O2 partial pressure declines below its resting value, it does so on the steep part of the oxygen equilibrium curve (FiguRE 24.6). Consequently, relatively small decreases in the venous O2 partial pressure result in relatively large increases in the yield of O2 from the blood. This point is illustrated in Figure 24.6. For discussing the figure, recall first that at rest, a drop in partial pressure from an arterial value of 12.0–12.7 kPa (90–95 mm Hg) to the resting venous value of 5.3 kPa (40 mm Hg)—a total drop of 6.7–7.4 kPa (50–55 mm Hg)—causes release of about 5 mL of O2 from each 100 mL of blood. As the figure shows, a further drop of just 2 kPa (15 mm Hg) to a venous partial pressure of 3.3 kPa (25 mm Hg) causes the blood to release another 5 mL of O2 from each 100 mL of blood, thus doubling the O2 yield. Moreover, a still further drop of just 1.3 kPa (10 mm Hg) to a venous partial pressure of 2 kPa (15 mm Hg) triples the yield of O2 from the blood! This steep release of O2 is a consequence of the binding characteristics of the hemoglobin molecule.

Arrows show the drop in blood O2 concentration as blood from the lungs flows through the systemic tissues. Each 100 mL of blood yields much more O2 during vigorous exercise (right arrow) than during rest (left arrow), because oxygenation in the lungs remains similar but deoxygenation in the systemic tissues is increased.

to yield more O2 in its passage through the tissue, and therefore

the venous partial pressure will decline. Similarly, an increase in

0 4 8 12 16kPa Partial pressure of O2 in blood

FiguRE 24.5 Oxygen delivery by human blood at rest and during vigorous exercise The oxygen equilibrium curve shown is that for human arterial blood (see Figure 24.4B).The thickened, shad- ed areas on the curve show representative ranges of blood O2 con- centration and O2 partial pressure in the lungs (blue), the systemic tissues during rest (green), and the systemic tissues during vigorous exercise (red).The vertical purple arrows to the right show how much O2 is delivered to the tissues by each 100 mL of blood during rest and exercise. All values are semi-quantitative; the intent of this diagram is conceptual rather than literal.Tissue values are mixed venous blood values. Effects of pH and other variables of the blood-hemoglobin milieu are not included.

in O2 partial pressure during circulation through all tissues com- bined. It does not necessarily reflect, however, the drop in partial pressure as the blood flows through any particular tissue; blood entering a particular tissue at a partial pressure of 12.7 kPa (95 mm Hg) might exit at a partial pressure that is either higher or lower than the mixed venous partial pressure. The mixed venous partial pressure is, in fact, a weighted average of the O2 partial pressures of blood leaving the various tissues. It is weighted according to the rate of blood flow through each tissue; tissues with high rates of blood flow influence the mixed venous partial pressure more than those with low rates of flow do.

THE DETERminAnTS OF A TiSSuE’S vEnOuS O2 PARTiAl PRES- SuRE The O2 partial pressure to which the blood falls in its passage through a particular tissue is not a static property of that

tissue. Instead, it is a dynamic and changing property. It depends

Hill Animal Physiology 4E
on the rate of blood flow through the tissue, the arterial O partial

HowgreatistheactualO deliveryduringexerciseinmam- 2

Sinauer Associates 2 pMreorsasleusreS,tutdhioe amount of hemoglobin per unit of blood volume,

mals? Over a wide range of exercise states, the O2 partial pressure of blood leaving the working skeletal muscles is about 2.7 kPa (20 mm Hg) in humans and also in several other species on which measurements have been made. At this value, the oxygen utiliza-

Figure 24.05 12-10-15

and the tissue’s rate of O2 consumption. To illustrate, if the rate of blood flow through a tissue decreases while all the other relevant factors remain unchanged, each unit of blood volume will have

the rate of O consumption by a tissue will cause a decrease in the 2

tissue’s venous partial pressure. The venous partial pressures nor- mally seen in people at rest result from the set of conditions that ordinarily prevail at rest.

Systemic tissues at rest

Lungs Rest

Exercise

Systemic tissues during exercise

O2 concentration of blood (mL O2/100 mL)

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When the blood starts at a high partial pressure of O2, its partial pressure must fall greatly (green arrow) for 5 mL of O2

to be released from 100 mL of blood (red arrow).

However, when the blood is already at a lowered partial pressure, its partial pressure must fall only a little (green arrow) for

5 mL of O2 to be released from 100 mL of blood (red arrow).

120 mm Hg

kPa (16–20 mm Hg) in humans, even during strenuous work, because blood from the exercising muscles mixes in the great veins with blood from other parts of the body in which O2 utilization is not so great. The whole-body oxygen utilization coefficient therefore rises to a peak of about 60%–75% during exercise—indicating that 2.5–3.0 times more O2 is extracted from each volume of blood than is extracted at rest, as illus- trated in Figure 24.5. In average young people, the rate of blood circulation can be increased to 4–4.5 times the resting level. These values, taken together, show that the total rate of O2 delivery by the circulatory system can increase to 10–13 times the resting rate. Trained athletes often achieve still higher O

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the rate at which a person’s heart can pump blood.

THE “mOlECulAR DESign” OF HumAn HEmOglOBin

We have seen in this section that (1) human hemoglobin is nearly saturated at the O2 partial pressures that are maintained in the lungs by breathing; (2) the oxygen equilibrium curve of hemoglobin is nearly flat at pulmonary O2 partial pressures, so that high oxygenation is ensured regardless of variation in pulmonary function; and (3) the oxygen equilibrium curve is shaped in such a way that 90% of the O2 bound to hemo- globin can be released for use at blood partial pressures that

0 4 8 12 16kPa Partial pressure of O2 in blood

FiguRE 24.6 As the O2 partial pressure of blood falls, less and less of a drop in partial pressure is required to cause unloading of 5 ml of O2 from each 100 ml of blood Each green horizontal arrow depicts the drop in O2 partial pressure required to cause the unloading depicted by the red vertical arrow to its right.The oxygen equilibrium curve shown is that for human arterial blood (see Figure 24.4B).

tion coefficient for blood flowing through the working skeletal muscles is about 65% (as compared with 25% at rest). We have said that a partial pressure of about 2.7 kPa (20 mm Hg) prevails in the blood leaving the muscles over a wide range of exercise states; that is, even as the muscles work harder and demand more O2, in this range little change occurs in their venous partial pressure or in the amount of O2 they obtain from each unit of blood volume. This stability of venous partial pressure occurs because the rate of blood flow to the muscles is adjusted: As the O2 demand of the muscles rises, their rate of blood flow increases in parallel, enabling them to draw O2 from an enhanced volume of blood per unit of time. Of course, the rate of blood flow cannot increase indefinitely. Once it is maximized, further increases in a muscle’s intensity of work result in further decreases in the venous O2 partial pressure. Indeed, during extreme exertion, the O2 partial pressure of blood leaving some muscles may fall close to zero, signifying virtually complete deoxygenation of the blood, corresponding to 100% O2 utilization.

As the O2 partial pressure of blood in the systemic capillaries

declines, there is a risk that the rate of O2 diffusion from the blood to

the mitochondria will become too low to support aerobic catabolism.

The venous O2 partial pressure below which aerobic catabolism

becomes impaired is known as the critical venous O2 partial

pressure. It is approximately 1.3 kPa (10 mm Hg) in mammalian

muscles. As we have seen, the rate of blood flow through muscles

is usually increased sufficiently to maintain the venous O2 partial

pressure above this critical level over a wide range of exercise states.

Human hemoglobin yields about 90% of its O2 before the venous

partial pressure falls below the critical level (see Figure 24.4A). In

Hill Animal Physiology 4E
this respect we see once more that the O2-binding properties of

during exercise. Although blood draining active muscles may be rather thoroughly deoxygenated during heavy exercise, the partial pressure of mixed venous blood generally does not fall below 2.1–2.7

are compatible with full mitochondrial function. These functional properties of human hemoglobin are consequences of its chemical structure, and its normal chemical structure is but one of thousands of possible structures. Many physiologists have concluded that the human hemoglobin molecule provides a particularly convincing example of “evolutionary molecular design.” Natural selection has produced a molecule with functional properties that are integrated in strikingly harmonious ways with the attributes of the organs that provide O2 to the blood and draw O2 from the blood.

A set of general principles helps elucidate O transport by respiratory pigments 2

From our study of hemoglobin function in people, we can state four key principles that are useful for understanding the function of blood respiratory pigments in general:

1. To determine the extent of pigment oxygenation, ask first: What are the blood O2 partial pressures established in the breathing organs? Then examine the oxygen equilibrium curve to determine the extent of pigment oxygenation at those partial pressures.

2. To determine the extent of pigment deoxygenation in systemic tissues, start by acquiring information on blood O2 partial pressures in those tissues. The mixed venous O2 partial pressure is a useful and easily measured indicator, although one must remember that it does not necessarily provide information on O2 release in any particular tissue. After the O2 partial pressure in the systemic tissues
has been measured or estimated, examine the oxygen equilibrium curve to determine the extent of pigment deoxygenation in the systemic tissues.

3. To compute circulatory O2 delivery, the rate of blood flow is as important as the yield of O2 per unit of blood volume,

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hemoglobin are closely integrated with other physiological features.

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Figure 24.0L6et’s0n1o-1w9-l1o6ok briefly at whole-body O2 utilization and O2 delivery

Transport of Oxygen and Carbon Dioxide in Body Fluids 645

2 delivery rates, principally because endurance training increases

O2 concentration of blood (mL O2/100 mL)

646 Chapter 24

(A) The oxygen equilibrium curve for human myoglobin

because O2 delivery is the product of flow rate and O2 yield per unit of volume. Complexity is introduced by the fact that these two factors are not independent: The rate
of blood flow helps to determine the venous O2 partial pressure and thus the yield of O2 per unit of blood volume.

4. The function of the O2-transport system is strongly affected by exercise. Full understanding of the function of an O2-transport system requires that animals be studied over a range of physiological conditions.

The shape of the oxygen equilibrium curve depends on O2-binding site cooperativity

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What determines the shape of the oxygen equilibrium curve? As
we explore this question, vivid parallels to principles we addressed
in the study of enzymes will again be evident. In Chapter 2 (see
page 46), we saw that when the catalytic sites of a particular en-
zyme function independently of each other, a hyperbolic relation
exists between enzyme activity and substrate concentration; when
the sites exhibit cooperativity, however, a sigmoid relation occurs. 80 Similarly, when the O2-binding sites of a respiratory pigment func-

tion independently, the oxygen equilibrium curve is hyperbolic, but 60 when they exhibit cooperativity, a sigmoid curve results.

Hyperbolic oxygen equilibrium curves are exemplified by the
vertebrate myoglobins (FiguRE 24.7A). The vertebrate (and most 40 invertebrate) myoglobins contain just one O2-binding site (heme)
per molecule. Thus their O2-binding sites function independently 20 of each other, and the chemical reaction between a myoglobin and
O2 can be written simply as

Mb + O2 ~ MbO2
where Mb is a molecule of deoxymyoglobin and MbO

(24.1)

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The pigment that shows no cooperativity requires a very low O2 partial pressure of 5 mm Hg (0.7 kPa) to release 80% of its O2, but...

...the pigment that exhibits cooperativity unloads 80% of its O2 at a much higher O2 partial pressure

60 80 100 120 mm Hg

is one of oxymyoglobin. According to the principles of mass action (see page 50), increasing the partial pressure (and thus the chemical potential) of O2 will shift this reaction to the right, increasing myoglobin oxygenation. Mass-action principles applied to such a simple chemical reaction also predict that the fraction of myoglobin molecules oxygenated will increase as a hyperbolic function of the

0 4 8 12 16kPa Partial pressure of O2

FiguRE 24.7 Respiratory pigments display hyperbolic or sig- moid oxygen equilibrium curves depending on whether they exhibit cooperativity in O2 binding (A) The hyperbolic oxygen equilibrium curve of human myoglobin—a pigment that exhibits no cooperativity—compared with the sigmoid curve of human blood hemoglobin—a pigment that displays cooperativity. Both curves were determined under similar conditions: 38°C, pH 7.40. (B) Comparison of oxygen equilibrium curves for two hypothetical pigments that reach saturation at about the same O2 partial pressure, but differ in whether they exhibit cooperativity. (A after Roughton 1964.)

with numerous O2-binding sites. All the curves are sigmoid to some

degree, indicating that intramolecular cooperativity occurs in all

cases. The extent of cooperativity, which varies from one respiratory

pigment to another, is usually expressed using a mathematical index

called the Hill coefficient (n), named after A. V. Hill (1886–1977),

a Nobel laureate. The coefficient is 1.0 for pigments that show no

cooperativity (e.g., myoglobins) and reaches 6 or more in some

Hill Animal Physiology 4E 10 high-molecular-weight pigments with very high cooperativity.

cooperativity on the oxygenation (loading) of respiratory pigments,

O2 partial pressure, as is observed (see Figure 24.7A).
Because vertebrate blood hemoglobins have four O2-binding sites within each molecule, the opportunity exists for cooperativity. In fact, positive cooperativity occurs in these hemoglobins: Binding of O2 at one or two of the O2-binding sites on a molecule of blood hemoglobin alters the conformation of the molecule in ways that enhance the affinity of the remaining sites for O2, meaning that a partially oxygenated molecule is more likely than an entirely deoxygenated one to bind additional O2.9 The consequence is a sigmoid oxygen equilibrium curve, exhibiting a particularly steep relation between O2 binding and O2 partial pressure in the mid-range of O2 partial pressures. FiguRE 24.8 presents oxygen equilibrium curves for the blood of 11 animal species, including 6 vertebrates that have four-unit hemoglobins and 5 invertebrates that have high-molecular-weight hemoglobins or hemocyanins

9 Because the four O2-binding sites are located within the four different protein subunits of the hemoglobin tetramer, the cooperativity displayed by the tetramer is often termed subunit interaction. It used to be termed heme–heme interaction, but this term has been dropped because the interaction between the O2-binding sites is indirect, not directly between one heme and another.

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0 4 8 12 16kPa Partial pressure of O2

(B) Hypothetical respiratory pigments that differ in cooperativity

100

Although in the last paragraph we emphasized the effect of

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Figure 24.07 12-10-15

cooperativity also affects deoxygenation (unloading). During de- oxygenation of a molecule that exhibits cooperativity, removal of O2 from some of the O2-binding sites tends to decrease the affinity of the remaining sites for O2, thereby promoting even further deoxygenation. 10

Mammalian hemoglobins exhibit values of 2.4–3.0.

No cooperativity

Cooperativity

of 22 mm Hg (3 kPa).

Percent saturation (%) Percent saturation (%)

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at which 50% 100 oxygen saturation

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FiguRE 24.9 How to measure P50

for full loading and that conversely unload substantial amounts of O2 at relatively high partial pressures are said to have a relatively low affinity for O2. Pigments that load fully at low partial pressures and consequently also require low partial pressures for substantial unloading are said to have a relatively high affinity for O2. Affinity for O2 is an inverse function of the O2 partial pressure required for loading: The higher the O2 partial pressure required to load a pig- ment, the lower is the pigment’s affinity for O2. The hemoglobins of humans and carp (see Figure 24.8) provide examples of pigments that differ in their affinity for O2. Human hemoglobin requires a far higher O2 partial pressure to become saturated than carp hemoglobin, indicating that the human hemoglobin combines less readily with O2 and has a lower affinity.

A convenient index of O2 affinity is P50 (pronounced “P fifty”),

defined to be the partial pressure of O2 at which a pigment is 50%

saturated. FiguRE 24.9 shows how P50 is measured. With Figure

0 4 8 12 kPa Partial pressure of O2 in blood

FiguRE 24.8 A diversity of blood oxygen equilibrium
curves The blood oxygen equilibrium curves of 11 animal species vary in two ways. First, they vary in shape, a property that reflects the different molecular forms of the respiratory pigments in different spe- cies. Second, they vary in height, a property that reflects how much respiratory pigment is present per unit of blood volume (oxygen- carrying capacity). Species: bullfrog, Rana catesbeiana (sometimes called Lithobates catesbeianus); carp, Cyprinus carpio; common earthworm, the nightcrawler Lumbricus terrestris; giant earthworm, the 1-m-long South American earthworm Glossoscolex giganteus; iguana, Iguana iguana; lugworm, the seacoast annelid Arenicola sp.; mackerel, Scomber scombrus; octopus, the giant octopus Enter- octopus dofleini of the North American Pacific coast; spiny lobster, Panulirus interruptus; Weddell seal, Leptonychotes weddelli. (After Hill and Wyse 1989.)

11
FiguRE 24.7B shows the consequences, using two hypothetical

pigments that are similar in the O2 partial pressure at which they become saturated, but differ in that one exhibits cooperativity whereas the other does not. If we assume that both pigments are initially fully oxygenated and ask how they behave during deoxygenation, it is clear

Hill Animal Physiology 4E
that in the mid-range of O2 partial pressures, the pigment showing

Respiratory pigments exhibit a wide range of affinities for O2

The respiratory pigments of various animals vary widely in how readily they combine with O2, a property known as their affinity for O2. Pigments that require relatively high O2 partial pressures

11 Figure 24.6 also does so.

24.9 in mind, a glance at Figure 24.8 reveals that human hemoglo-

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cooperativity deoxygenates more readily, giving up more of its O2 at

...the O2 partial pressure needed to saturate is higher, and...

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anygivenO partialpressure.Inasentence,whetheramoleculeofa Figure 24.08 2 12-10-15

respiratory pigment is loading or unloading, cooperativity enhances the responsiveness of the process to changes in the O2 partial pressure within the mid-range of partial pressures.

bin has a much higher P50 (≅ 3.5 kPa in arterial blood) than carp

hemoglobin (≅ 0.7 kPa). Affinity and P are inversely related: As P

increases, O affinity decreases. 50 50 2

In the jargon of respiratory-pigment physiology, lowering the O2 affinity is said to “shift the oxygen equilibrium curve to the right.” To explain, FiguRE 24.10 shows that a rightward shift (a shift from the blue to the red curve) reflects a higher P50 and therefore a lower O2 affinity. Raising the O2 affinity (decreasing the P50)—as would occur by shifting from the red to the blue curve—is said to “shift the curve to the left.”

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Hill A Sinaue Morale Figure

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A shift to the right means...

Partial pressure of O2

FiguRE 24.10 A “shift to the right” Such a shift reflects de- creased O2 affinity.

Weddell seal

Giant earthwor

Carp

Human

Mackerel

Lugworm

Common earthworm

Iguana

Bullfrog

Octopus Spiny lobster

P ...the P50 is

nimal Ph50ysioPlogy 4E r Associates 50
s Studio

24.09 12-10-15

higher. Thus... ...O2 affinity is lower.

Percent saturation (%)

O2 concentration of blood (mL O2/100 mL)

Percent saturation (%)

648 Chapter 24
(A) Human hemoglobin at various pH levels

Christian Bohr (1855–1911), the prominent Danish physiologist (and father of Nobel laureate Niels Bohr) who led the discovery of the effect in 1904. Part of the reason that an increase in CO2 partial pressure causes such a shift is that the pH of a solution tends to decline as its CO2 partial pressure is increased.13 However, CO2 also exerts a direct negative effect on the O affinities of some

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Affinity for O2 decreases as blood pH decreases. As affinity decreases, the oxygen equilibrium curve shifts to the right.

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respiratory pigments, such as the blood hemoglobins of humans

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and other mammals. Recognizing that protons (H+ ions) and CO2 itself can exert independent affinity-lowering effects, modern workers often distinguish two types of Bohr effects: a fixed-acid Bohr effect—which results from influences of the proton (H+) concentration on respiratory-pigment molecules—and a CO2 Bohr effect—which results from the immediate influences of increased CO2 partial pressure.

Species that show these effects vary widely in the magnitudes of the effects. One reason is that Bohr effects have probably evolved several times independently and thus have a different molecular basis in some animals than others. Even species with the same molecular mechanism often vary widely in details.

Protons exert their effects on O2 affinity by combining with pigment molecules. Referring to hemoglobin (Hb) as a specific example, we can write the following strictly conceptual equation to summarize the effects of protons on O2 affinity (the equation does not reflect the true stoichiometry of the reaction):

HbO2 + H+ ~ HbH+ + O2 (24.2)

Increasing the H+ concentration tends to increase the combination of Hb with H+, thus shifting the chemical reaction in Equation 24.2 to the right and favoring dissociation of O2. The H+ ions bind at sites on the hemoglobin molecules (e.g., at histidine residues) different from the O2-binding sites. Thus H+ acts as an allosteric modulator of O2 binding. CO2 also combines chemically with pigment mol- ecules and functions as an allosteric modulator in cases in which it exerts direct effects on affinity.

The Bohr effect often has adaptive consequences for O2 delivery. The CO2 partial pressure is generally higher, and the pH is generally lower, in the systemic tissues than in the lungs or gills. Because of this, a respiratory pigment that displays a Bohr effect shifts to lower O2 affinity each time the blood enters the systemic tissues and reverts back to higher O2 affinity each time the blood returns to the breathing organs. The shift to lower affinity in the systemic tissues promotes release of O2 because it facilitates deoxygenation. Conversely, the shift back to higher affinity in the breathing organs promotes uptake of O2 by facilitating oxygenation. FiguRE 24.12 illustrates the net effect of this shifting back and forth between two oxygen equilibrium curves as the blood flows between the breathing organs and systemic tissues. At any given O2 partial pressures in the arterial and venous blood, more O2 is delivered to the systemic tissues than would be if the pigment followed just one or the other equilibrium curve alone.

During exercise, the CO2 partial pressure in the systemic tissues often rises above that prevailing during rest because of the increased production of CO2. Furthermore, the pH in the systemic tissues often falls below the resting pH, not only because of the elevated

13 As already noted in Chapter 23, CO2 has been aptly termed a “gaseous acid” because it reacts with H2O to produce H+. The chemistry of these reactions is presented at length later in this chapter.

0 4 8 12 16 20 kPa Partial pressure of O2 in blood

(B) Dog hemoglobin at various CO2 partial pressures

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Affinity for O2 decreases as the CO2 partial pressure of
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0 4 8 12 16 20 kPa Partial pressure of O2 in blood

pH decreases or CO2 partial pressure increases (A) Oxy- gen equilibrium curves of human hemoglobin at three different pHs at 38°C. In resting humans, the normal pH of arterial blood is about 7.4, whereas that of mixed venous blood is about 0.04 unit less. (B) Oxygen equilibrium curves of dog hemoglobin at five different CO2 partial pressures at 38°C.The data in part (B) are from the original work of Bohr and his coworkers. (After Roughton 1964.)

The Bohr effect: Oxygen affinity depends on the

FiguRE 24.11 The Bohr effect: Affinity for O decreases as 2

partial pressure of CO

2

and the pH

In a body fluid or tissue containing a respiratory pigment, a decrease

in the pH or an increase in the CO2 partial pressure often causes

the O2 affinity of the respiratory pigment to decrease, thus shifting

the oxygen equilibrium curve to the right. This effect, illustrated

Hill Animal Physiology 4E
for the blood hemoglobins of humans and dogs in FiguRE 24.11,

Sinauer Associates 12
is known as the Bohr effect or Bohr shift, in commemoration of

Morales Studio
Figure 24.11 12-11-15

12 In unusual cases, such as some species of molluscs and spiders, Bohr effects opposite to the usual direction, termed reverse Bohr effects, are observed.

pH = 7.6

pH = 7.4

pH = 7.2

0.7 kPa

5 mm Hg

1.3 10

CO

partial

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2 pressures

5.3 40

10.7 80

Percent saturation (%) Percent saturation (%)

This is the assumed venous O2 partial pressure.

8 kPa

15 12 9 6 3 0

0 0

20

80 mm Hg

Shift between A and V (7.2 mL)

Oxygen delivery to the tissues is greater in the presence of the Bohr shift (shift from curve A to curve V )...

...than it would be if the blood were to adhere exclusively to curve A or curve V.

4
Partial pressure of O2 in blood

CO2 partial pressure but also because acid metabolites—such as lactic acid—often accumulate during exercise. These changes often augment the Bohr shift during exercise, thereby enhancing O2 delivery to the active tissues.

Now it will be clear why we indicated earlier in this chapter that refinements would ultimately be needed to our initial analysis of O2 delivery in humans. We based our earlier analysis on the arterial oxygen equilibrium curve alone (see Figure 24.4), whereas in reality, Bohr shifts occur as blood flows between the lungs and systemic tissues. In humans at rest, venous blood is slightly more acidic (pH 7.36) than arterial blood (pH 7.40). Moreover, the CO2 partial pressure is higher in mixed venous blood than in arterial blood: about 6.1 kPa (46 mm Hg) in venous blood and 5.3 kPa (40 mm Hg) in arterial. Looking at Figure 24.11, you can see that these differences in pH and CO2 partial pressure are sufficient to cause small but significant Bohr shifts of the oxygen equilibrium curve as blood flows between the lungs and systemic tissues.

To fully understand respiratory-pigment function, it is important

that, before closing this section, we consider not only how pH can

affect oxygenation, but also how oxygenation can affect pH. Let’s

return to the conceptual equation, Equation 24.2, that describes

the reaction of H+ ions with respiratory pigments (assuming that

a fixed-acid Bohr effect exists). Earlier we stressed one perspective

on this equation; namely, that an increase in H+ concentration will

push the chemical reaction to the right, decreasing the tendency

of pigment molecules to bind to O2. Now we also stress that the

The Root effect: In unusual cases, CO2 and pH dramatically affect the oxygen-carrying capacity of the respiratory pigment

In some types of animals, because of distinctive properties of their respiratory pigments, an increase in the CO2 partial pres- sure or a decrease in the pH of the blood not only causes a Bohr effect, but also reduces the amount of O2 the respiratory pigment binds when saturated.14 The reduction in the amount of O2 bound to the pigment at saturation (FIguRe 24.13) is termed the Root effect, after its discoverer. Root effects of sizable magnitude are not common. Among vertebrates, they are observed only in fish, principally teleost fish. Some molluscs also show either normal or reversed Root effects.

Root effects provide a mechanism by which the O2 partial pres-

sure of even well-oxygenated blood can be dramatically increased

under the control of blood pH. To see this, consider the hemoglobin

in the blood of eels when it is fully loaded with O2 (see Figure

24.13).15 At a pH of 7.54, the hemoglobin is chemically combined

with about 12.6 mL of O2 per 100 mL of blood. Acidification to a pH

of 7.35 lowers the O2-binding capacity of the hemoglobin because

of the Root effect, so that the hemoglobin can chemically combine

with only about 9.4 mL O2/100 mL. In this way O2 is forced off the

hemoglobin by the change of pH. The acidification from pH 7.54 to

7.35 forces the eel hemoglobin to unload 3.2 mL of O2 into each

100 mL of blood! The O2 released goes into blood solution; it has

no other immediate place to go. By dissolving, it dramatically elevates

the blood O partial pressure. 2

The Root effect is employed in various species of teleost fish to help create high O2 partial pressures in two regions of the body: the swim bladder and the eyes. In both types of organs, the pH of well-oxygenated blood is lowered by a tissue-specific addition of lactic acid, which induces a rise in the blood O2 partial pressure

14 Some modern authorities view the Root effect as an exaggerated Bohr effect.

15

equation has a complementary and equally important property:

Hill Animal Physiology 4E
Removal of O from pigment molecules will pull the chemical

Sinauer Associates 2 +

Morales Srteuadciotion to the right, causing the pigment molecules to take up H

Figure 24.12 12-11-15

inbloodpH—causedbythemetabolicadditionofCO andH+. 2

from their surroundings. When blood passes through the systemic tissues, metabolism tends to increase the H+ concentration of the blood solution. Simultaneously, however, because of the diffusion of O2 out of the blood, respiratory-pigment molecules unload O2 and thus bind with H+. This removal of free H+ from the blood, induced by the deoxygenation of the pigment molecules, limits the increase in the blood concentration of H+—and the decrease

Although Figure 24.13 serves as a useful visual guide, the insight it provides into hemoglobin function is qualitative, not quantitative, because the O2 concentrations shown include dissolved O2.

Transport of Oxygen and Carbon Dioxide in Body Fluids 649

The vertical arrows show the drop in blood O2 concentration as blood from the breathing organs flows through the systemic tissues.

FIguRe 24.12 The Bohr effect typi- cally enhances O2 delivery in an animal The diagram shows oxygen equilibrium curves for arterial blood (A) and venous blood (V) in a hypotheti- cal animal.The venous blood displays a reduced O2 affinity because its CO2 partial pressure and H+ concentra-

tion are higher than those in arterial blood.The magnitude of this Bohr shift is exaggerated for clarity.The three bold arrows show unloading under three different assumptions.The top of each arrow is the O2 concentration of blood as it leaves the breathing organs; the bottom is the O2 concentration of the blood as it leaves the systemic tissues. The numeric values next to the arrows are the volumes of O2 delivered per 100 mL of blood.

This is the assumed arterial O2 partial pressure.

V alone (6.5 mL)

A alone (3.3 mL)

A

V

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O2 concentration of blood (mL O2/100 mL)

650

Chapter 24

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6.99

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0 4 8 12 kPa Partial pressure of O2 in blood

FiguRE 24.14 An increase in temperature typically causes
a decrease in O2 affinity Oxygen equilibrium curves are shown for human blood at six different temperatures, with pH held constant at 7.4.These results show the pure effect of changes in temperature because of the constancy maintained in pH.The results,however, tend to understate the effects of temperature in many real-life situa- tions because when the pH is not artificially controlled, a rise in blood temperature typically induces a decrease in blood pH, as discussed later in this chapter (see Figure 24.24), meaning that the immedi-
ate effects of temperature are often reinforced by thermally induced fixed-acid Bohr effects.The CO2 partial pressure was held constant during the studies shown. (After Reeves 1980.)

temperature effects may become a problem in the hypothermic limbs of mammals in Arctic climates—a matter addressed in BOx 24.3 in relation to recent studies of a resurrected ancient protein, the hemoglobin of the woolly mammoth.

Organic modulators often exert chronic effects on oxygen affinity

Organic compounds synthesized by metabolism often play major

roles as allosteric modulators of the function of respiratory pigments.

In vertebrates, the principal compounds acting in this role are or-

ganophosphate compounds within the red blood cells, which affect

hemoglobin O2 affinity. The organophosphate of chief importance

in most mammals, including humans, is 2,3-bisphosphoglycer-

0 4 8 12 16 20 kPa Partial pressure of O2 in blood

FiguRE 24.13 The Root effect in eels: Acidification lowers the oxygen-carrying capacity of hemoglobin Oxygen equilib- rium curves are shown for the whole blood of eels (Anguilla vulgaris) at six different pH levels (at 14°C). Saturation of hemoglobin is indi- cated when the slope of an oxygen equilibrium curve parallels the slope of the dissolved O2 line. Experiments on some fish have shown that O2 binding by hemoglobin is reduced at low pH even when the hemoglobin is exposed to an O2 partial pressure of 140 atmospheres (atm) (14,000 kPa)! (After Steen 1963.)

because of the Root effect. Moreover, in both types of organs, this rise in the blood O2 partial pressure is amplified by a countercurrent vascular arrangement (a rete mirabile) that favors multiplication of the initial effect.16 The creation of a high O2 partial pressure helps inflate the swim bladder in many fish (swim-bladder gas is often principally O2). The retinas of some fish are so poorly vascularized that they require a high surrounding O2 partial pressure to acquire enough O2 to function properly. Recently, a convincing case has been made that, in the course of evolution, the first role of the Root effect in fish was oxygenation of the retina. Later, at least four different lines of fish independently evolved the use of the Root effect in O2 secretion to inflate the swim bladder.

Thermal effects: Oxygen affinity depends on tissue temperature

ate, which is synthesized in red blood cells from intermediates of Hill Animal Physiology 4E

The O affinity of respiratory pigments is often inversely depen- 2

glycolysis. This compound is sometimes called BPg or 2,3-BPg, Sinauer Associates

dent on temperature (FiguRE 24.14). Increases in temperature de- Animal Physiology 4E

Morales Studio

Hill

SinaucerreAasseoacifaftiensity, whereas decreases in temperature increase affinity

2,3-DPg (standing for 2,3-diphosphoglycerate) is used. The effect Figure 24.14 12-11-15

of 2,3-DPG is to reduce the O2 affinity (raise the P50) of the hemo- globin molecules with which it binds. As shown by the black line in FiguRE 24.15, hemoglobin O2 affinity is therefore a function of the 2,3-DPG concentration. The hemoglobin of humans and most other mammals is continuously exposed to and modulated by 2,3-DPG within the red blood cells. Thus, as stressed in Figure 24.15, the “normal” O2 affinity of human hemoglobin in the red blood cells is in part a consequence of modulation by a “normal” 2,3-DPG concentration within the cells.17

17 Some mammals have hemoglobins that are not modulated by organophosphate compounds under ordinary physiological conditions. Included are some ruminants, cats, civets, and related species. Their hemoglobins, within the red blood cells, display functionally appropriate O2 affinities without 2,3-DPG, and the red blood cells of adult animals of these types usually contain little 2,3-DPG.

Morales Studio

(changes in temperature only rarely affect the O2 content of blood

Figure 24.13 12-11-15

at saturation, however). When humans or other mammals exercise, if the blood temperature in their exercising muscles exceeds the temperature in their lungs, thermal shifts in affinity will enhance O2 delivery to the muscles in a manner much like that already described for the Bohr effect (see Figure 24.12). In total, therefore, unloading of O2 to the exercising muscles will be promoted in a concerted manner by both a temperature effect and a Bohr effect, both of which independently tend to decrease the O2 affinity of the respiratory pigment (and thereby facilitate O2 unloading) when the blood passes through the muscles. Conversely to this happy state,

16 The process of countercurrent multiplication is explained, in a different context, in Chapter 29 (see pages 792–793).

but more commonly, for historical reasons, the abbreviated name

O2 concentration of blood (mL O2/100 mL)

Percent saturation (%)

mm Hg 45

35

25

kPa shows the normal P50 6

FiguRE 24.15 The normal P50 of human hemoglobin within red blood cells depends on a normal intracellular concentration of 2,3-DPg There is usually about one 2,3-DPG molecule per hemo- globin molecule in human red blood cells.The temperature (37°C),CO2 partial pressure (5.3 kPa), and extracellular pH (7.4) were held constant during the measurements presented here. (After Duhm 1971.)

The blue shaded area

values of hemoglobin within red blood cells.

15
0 4 8 12 16 20 24

Concentration of 2,3-DPG (μmole/g erythrocytes)

4

2

organophosphates act as modulators in these other groups, their effect is to lower O2 affinity.

Chronic changes in the concentration of organophosphate modulators in red blood cells serve as mechanisms of acclimation or acclimatization in many vertebrates. People suffering from anemia, to mention one example, often exhibit a chronic increase in the concentration of 2,3-DPG in their red blood cells; the O2 affinity of their hemoglobin is thereby lowered by comparison with the usual affinity. The resulting shift to the right in their oxygen equilibrium curve is not great enough to cause any substantial impairment of O2 loading in their lungs, but it significantly facili- tates O2 unloading in their systemic tissues (FiguRE 24.16). Thus each molecule of hemoglobin, on average, delivers more O2 from the lungs to the systemic tissues during each passage through the circulatory system. In anemic people, this effect helps offset the disadvantage of having a reduced amount of hemoglobin per unit of blood volume.

In nonmammalian vertebrates, modulation of hemoglobin O2 affinity by red blood cell organophosphates is also very com- mon, although the specific phosphate compounds that bind with and allosterically affect hemoglobin vary from one taxonomic group to another and usually do not include 2,3-DPG. ATP and guanosine triphosphate (GTP) are generally the principal organo- phosphate modulators in fish. In birds, inositol pentaphosphate (IPP) and ATP are especially important. As in mammals, when

Transport of Oxygen and Carbon Dioxide in Body Fluids 651

Relation between P50 and 2,3-DPG concentration

The green shaded area marks the range of 2,3-DPG concentrations typically observed in red blood cells.

P50

652 Chapter 24

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...each 100 mL
of blood delivers more O2 from the breathing organs to the systemic tissues during each passage through the circulatory system when the affinity is reduced.

100 mm Hg

0 4 8 12 kPa Partial pressure of O2 in blood

FiguRE 24.16 A decrease in the O2 affinity of hemoglobin can aid O2 delivery to the systemic tissues when the O2 partial pressure in the breathing organs remains high Two human oxygen equilibrium curves, representing normal and reduced O2 affinities, are shown.The loading O2 partial pressure in the lungs is assumed to be 12.7 kPa (95 mm Hg), and the unloading O2 partial pressure in the systemic tissues is assumed to be 5.3 kPa (40 mm Hg). The green vertical arrows show the changes in percent saturation at these two partial pressures caused by the shift from normal affinity to reduced affinity (for simplicity and clarity, other effects on affinity, such as Bohr effects, are ignored, and the reduction in affinity is exagger- ated).The principles elucidated here apply to gill breathers as well as lung breathers.

The arthropod hemocyanins are well known to be modu- lated by organic compounds such as lactate ions, dopamine, and trimethylamine. In many crustaceans, for example, O2 affinity is elevated by increasing plasma concentrations of lactate ions (specifically l-lactate ions), which exert their effects by binding to specific allosteric sites on the hemocyanin molecules. When animals such as blue crabs (Callinectes sapidus) engage in exercise that produces lactic acid (see Chapter 8), the affinity-increasing effect of the lactate ions offsets the large affinity-decreasing effect of the acidification of their blood (Bohr effect), helping to ensure that their hemocyanin remains capable of fully loading with O2 in the gills.

The Functions of Respiratory

Pigments in Animals

It would be hard to exaggerate the diversity of functional proper- ties found among animal respiratory pigments. The oxygen affin- ity (P50) of respiratory pigments varies from less than 0.2 kPa to more than 7 kPa. Cooperativity (the Hill coefficient, n) varies from 1 to more than 6. The concentration of the respiratory pigment in an animal’s blood may be so low that the pigment merely doubles the oxygen-carrying capacity of the blood in comparison with the dissolved O2 concentration; alternatively, a pigment may be so concentrated that it allows blood to carry 80 times more O2 than can be dissolved. A respiratory pigment may or may not exhibit a Bohr effect or temperature effect. One pigment may be modulated by 2,3-DPG, another by ATP. With this diversity of properties, even when the respiratory pigments of various animals carry out a single function, they do so in a diversity of detailed ways.

Respiratory pigments, moreover, are presently known to carry out at least eight different functions—meaning that, overall, they have a very wide range of action. The functions are not mutually exclusive; often a single respiratory pigment carries out two or more functions simultaneously. Although we will cover only a few functions in any detail, all eight deserve recognition:

ill Animal Phinysoiolrogya4nE ic ions may also act as modulators inauer Associates

of respiratory pigments

orales Studio
igure 24.16 1C2-o1n1c-1e5ntrations of specific inorganic ions in blood cells or blood

plasma sometimes allosterically modulate the O2 affinity or other attributes of respiratory pigments. Recent research, for example, has revealed that in many ruminant mammals and certain bears, the concentration of Cl– in the red blood cells is a critical allosteric modulator of hemoglobin function. The divalent ions Ca2+ and Mg2+ are important allosteric modulators of hemocyanin in crustaceans. Blue crabs exposed to O2-poor waters, for example, increase their blood Ca2+ concentration, which raises the O2 affinity of their hemocyanin.

Percent saturation (%)

H S M F

1. Respiratory pigments in blood (or other circulating body fluids) typically aid the routine transport of O2 from the breathing organs to the systemic tissues. This is the function to which we have devoted most of our attention up to this point in the chapter.
2. Respiratory pigments in the blood of some invertebrates probably function primarily as O2 stores, rather than participating in routine O2 transport. The pigments that fit this description have very high O2 affinities. Consequently, they hold so tightly to O2 that they probably do not unload under routine conditions. Instead, they seem to release their O2 when animals face severe O2 shortages. In certain species of tube-dwelling marine worms, for example, O2 bound to a high-affinity blood hemoglobin is believed to be unloaded primarily during periods when the worms do not breathe, when their tissue O2 partial pressures fall very low.
3. Blood respiratory pigments often serve as major buffers
   of blood pH and thereby play key roles in blood acid–base regulation. As already mentioned (see Equation 24.2), this buffering is often of an “active” sort, in the sense that the affinity of the respiratory pigments for H+ changes as they unload and load O2. The pigments tend to remove H+ from solution as they become deoxygenated and release H+ into solution as they become oxygenated. We return to this topic later in this chapter (e.g., see Figure 24.23).
4. Blood respiratory pigments often play critical roles in CO2 transport, as we will also see later in the chapter.
5. Hemoglobins in the cytoplasm of muscle cells (myoglobins), or in the cells of other solid tissues, play two principal respiratory roles. First, they increase the rate of O2 diffusion through the cytoplasm of the cells, a phenomenon that in muscle cells is called myoglobin-facilitated O diffusion: At any given difference in O2 partial pressure between the blood capillaries and the mitochondria
   of the cells, O2 diffuses through the cytoplasm to the mitochondria faster if myoglobin is present. The second role played by hemoglobins within solid tissues is O2
   storage for the tissues. The myoglobin-bound O
   skeletal muscles, for example, can be called upon at the start of sudden, vigorous muscular work to help sustain aerobic ATP production while circulatory O2 delivery is still

and it transports S2– as well as O2 from the gills to the organ in which the bacteria live.

8. Finally, the fastest-breaking story in the contemporary study of respiratory pigments is the increasing recognition that at least in mammals, myoglobins are sometimes intimately involved in several tissue functions other than

O2 supply, especially the regulation of mitochondrial respiration, as addressed in BOx 24.4.

Patterns of circulatory O2 transport: The mammalian model is common but not universal

Circulatory O2 transport in most animals qualitatively follows the pattern we described earlier for mammals (see Figure 24.5). This pattern has several major features, which, for example, can be seen in the O2 transport physiology of rainbow trout (FiguRE 24.17). First, the blood respiratory pigment reaches near-saturation in the lungs or gills when the animals are living in well-aerated environ- ments. Second, the respiratory pigment yields just a modest fraction of its O2 to the systemic tissues during circulation at rest, mean- ing that venous blood in resting individuals is far from being fully deoxygenated. Third, the large resting venous O2 reserve is used (i.e., venous blood becomes more deoxygenated) during exercise or other states of heightened metabolism. Thus increased tissue O2 demands are met by increasing the amount of O2 delivered per unit of blood volume, as well as by increasing the rate of blood flow.

Squids and octopuses are important examples of animals that follow a different pattern of circulatory O2 transport, and thus

being accelerated to meet the heightened O

2

2

need.18

...the O2
delivery per
unit of blood volume increases about 2.5-fold

as trout swim faster.

1. Sometimes, respiratory pigments act as enzymes, not
   in carrying out their roles in O2 transport, but in
   other contexts. At least in mammals, for example, deoxymyoglobin and deoxyhemoglobin catalyze the local formation of the critically important signaling compound nitric oxide (NO) from nitrite in certain settings.
2. Respiratory pigments occasionally play nonrespiratory transport roles. In at least some species of worms that have symbiotic sulfur-oxidizing bacteria (see Figure 6.16), for example, the blood hemoglobin has sulfide-binding sites,

18 This role is discussed at length in Chapters 8 and 20.

Rest

50 60 70 80 90 100 Swimming speed (% maximum)

Transport of Oxygen and Carbon Dioxide in Body Fluids 653

store in

concentration in venous blood

2

The arterial blood remains nearly saturated at all levels of exertion. On the other hand...

decreases as swimming speed increases. Thus...

10 8 6 4 2 0

...the O 2

FiguRE 24.17 Blood O transport in rainbow trout in rela- 2

tion to exercise The lines show the average O2 concentration
of arterial and venous blood in trout (Oncorhynchus mykiss) at rest and swimming at various speeds in well-aerated water.The numbers above the arterial points show the average arterial percent satura- tion of the particular fish studied at each speed.As fish increase their speed from rest to maximum, they increase O2 delivery per unit of blood volume about 2.5-fold.Trout also increase their rate of circula- tion about 3-fold.Thus the trout increase the total rate of O2 delivery to their tissues about 7-fold. (After Jones and Randall 1978.)

97.0%

96.0%

99.7%

98.5%

98.8% Arterial O concentration

2

Venous O2 concentration

O2 concentration of blood
(mL O2/100 mL)

654 Chapter 24

Arterial values of percent O2 saturation, O2 partial pressure, and CO2 partial pressure normally fall within the dark purple area. On the other hand...

Partial pressure of CO2

0.4 3.2 0.05 0.4

6.0 mm Hg 0.8 kPa

FiguRE 24.18 Blood O2 delivery in an octopus: Even at rest, octopuses have almost no venous reserve The octopuses (En- teroctopus dofleini) studied were resting or only moderately active in well-aerated water.The three oxygen equilibrium curves correspond to three different blood CO2 partial pressures (i.e., the hemocyanin exhibits a Bohr effect).All the data obtained on arterial blood fall within the

dark purple area, whereas the data on venous blood fall within the light purple area.The two dots represent the approximate means for arterial and venous blood. (After Johansen and Lenfant 1966.)

illustrate that the pattern observed in mammals and fish is not universal. The squids and octopuses that have been studied have only a very small venous O2 reserve when they are at rest: Inactive individuals use 80%–90% of the O2 available in their arterial blood (FiguRE 24.18). Thus, when the animals exercise, they have little room to increase unloading of O2 from their hemocyanin, and they must meet their heightened O2 demands almost entirely by increasing their circulatory rates. This pattern places high demands on their hearts and constrains their ability to exercise, as we will see in more detail in Chapter 25 (see Box 25.3). The inherently small venous O2 reserve of the squids and octopuses also limits their ability to live in poorly aerated waters. If a squid or octopus ventures into O2-poor waters and consequently can’t oxygenate its arterial blood fully, it can’t compensate to any great degree (as a fish can) by enhancing the deoxygenation of its venous blood, because the venous blood is already highly deoxygenated even in aerated waters. Squids and octopuses are notoriously intolerant of low-O2 environments.

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Percent saturation (%)

Regardless of the exact pattern of circulatory O2 delivery an animal displays, the oxygen-carrying capacity of its blood—which depends on the amount of respiratory pigment per unit of blood volume—is a key determinant of how much O2 can be delivered to its tissues. As already seen in Figure 24.8, animals display a wide range of oxygen-carrying capacities. The range of known values in animals that have blood respiratory pigments is from about 30–40 mL O2/100 mL of blood in some diving mammals to just 1–2 mL O2/100 mL in many crustaceans and molluscs. Among vertebrates, a rough correlation exists between metabolic intensity and the oxygen-carrying capacity of the blood; mammals and birds usually have carrying capacities of 15–20 mL O2/100 mL, whereas fish, amphibians, and nonavian reptiles usually have less hemoglobin per unit of volume and have carrying capacities of 5–15 mL O2/100 mL. Active species of fish such as tunas and lamnid sharks tend to have higher oxygen-carrying capacities than do related sluggish species.

Animals with hemocyanin tend to have low oxygen-carrying capacities. Squids and octopuses exhibit the highest carrying capaci- ties known for hemocyanin-containing bloods, and their carrying capacities are just 2–5 mL O2/100 mL (at or below the lower end of the range for fish). Animals with hemocyanin—which is always dissolved in the blood plasma, not contained in blood cells—prob- ably cannot have much higher carrying capacities because the hemocyanin concentrations needed for higher capacities would make their blood too viscous to pump.

Individual animals can vary their oxygen-carrying capacity by raising or lowering the amount of respiratory pigment per unit of blood volume. The most common responses of this sort are long-term, occurring during acclimation or acclimatization to changed environments (to be discussed shortly). Some vertebrates, however, can acutely change their carrying capacity because they can remove red blood cells from their blood, store the cells, and quickly release them back into the blood. Horses, dogs, and some seals are well known to store massive quantities of red blood cells in their spleen when at rest. When the cells are needed during exercise, they are quickly released back into the blood under control of the sympathetic nervous system. Foxhounds, for example, can promptly increase their oxygen-carrying capacity from 16 to 23 mL O2/100 mL in this way.

at the expense of unloading of the blood hemoglobin. Thus the difference in affinity promotes transfer of O2 from the blood to the muscle cells.

Affinity relations also promote the transfer of O2 from mother to fetus across the placenta in placental mammals. Generalizing across species, the P50 of fetal blood is typically less than the P50 of maternal blood by 0.4–2.3 kPa (3–17 mm Hg). Because the fetal blood has a higher O2 affinity, it tends to oxygenate by drawing O2 from the maternal blood. The relatively high affinity of the fetal blood also means that it is able to become relatively well oxygenated even if the O2 partial pressure in the placenta is relatively low. Several specific mechanisms account for the differences in O2 affinity between fetal and maternal bloods in various species. In humans and other primates, the difference occurs because the chemical structures of the fetal and maternal hemoglobins are different, as mentioned at the beginning of this chapter. One key effect of these structural differences is that the fetal hemoglobin is less sensitive to 2,3-DPG; because 2,3-DPG lowers affinity, the diminished sensitivity of fetal hemoglobin to 2,3-DPG raises its O2 affinity. In some other species, such as dogs and rabbits, the hemoglobins in the fetus and mother are chemically the same; the reason the fetal affinity is higher is that fetal red blood cells have lower intracellular concentrations of 2,3-DPG than maternal red blood cells. In still other species of mammals, additional mechanisms of raising the fetal O2 af- finity are observed; ruminants, for example, have fetal forms of hemoglobin that are intrinsically higher in affinity than maternal hemoglobin, without 2,3-DPG modulation.

The relatively high O2 affinity of fetal hemoglobin is not necessarily the only factor that promotes O2 transfer from the mother’s blood to the blood of the fetus. An extremely interesting additional factor is that often the loss of CO2 from the fetal blood to the maternal blood induces a synchronous rise in fetal O2 affinity and fall in maternal O2 affinity because of Bohr effects in the two hemoglobins: Bohr effects that have these opposing but reinforcing consequences!

Evolutionary adaptation: Respiratory pigments are molecules positioned directly at the interface between animal and environment

A dramatic property of the respiratory pigments is that they are molecules that, in a way, actually form part of the interface be- tween an animal and its environment: They pick up O2 from the environment and deliver it to cells deep within tissues. Moreover, evolution has produced hundreds of different molecular forms of the respiratory pigments. Because of these considerations, the re- spiratory pigments have long been regarded as prime subjects for the study of evolutionary molecular adaptation.

Such studies have revealed that often species that have long histories of existence in low-O2 environments have evolved respira- tory pigments with higher O2 affinities than related species living in high-O2 environments. This common pattern is well illustrated by the fish in Figure 24.8 and by other fish: Carp and catfish, which often inhabit waters low in O2, have average P50 values of 0.1–0.7 kPa (1–5 mm Hg)—meaning their hemoglobins load particularly well at low O2 partial pressures—whereas mackerel and rainbow trout, which live in well-aerated waters, have far higher P50 values

Respiratory pigments within a single individual often display differences in O affinity that aid

successful O

2

transport 2

Two respiratory pigments often exist within one animal and pass O2 from one to the other. Most commonly, this occurs in animals that have myoglobins. In these animals, the blood respiratory pigment (hemoglobin or hemocyanin) and the myoglobin act as a sort of “O2 bucket brigade”: The blood pigment carries O2 from the lungs or gills to the muscles, and then passes the O2 to the myoglobin in the muscle cells. This process is typically aided by differences in O2 affinity. Specifically, the myoglobin typically has a higher O2 affinity—a lower P50—than the blood pigment; one can see in Figure 24.7A, for example, that the P50 of human myoglobin (about 0.8 kPa, 6 mm Hg) is far lower than that of hu- man blood hemoglobin (about 3.5 kPa, 27 mm Hg). The higher O2 affinity of the myoglobin means that it tends to load with O2

Transport of Oxygen and Carbon Dioxide in Body Fluids 655

656 Chapter 24
mm Hg kPa

5

35

30 4

unloading in the systemic tissues, it could potentially also diminish O2 delivery by interfering with O2 loading in the breathing organs. For sorting out these complexities, a crucial question is whether the O2 partial pressure in the breathing organs is high enough to cause full oxygenation. To explain, consider a case in which arterial O2 partial pressures are consistently high enough for respiratory pigments to be well oxygenated regardless of O2 affinity. In this case, the principal effect of low affinity is to promote unloading of O2 in the systemic tissues, which augments O2 delivery.20 You will recognize this argument. It is exactly why researchers think that small-bodied mammals living aboveground near sea level can benefit by evolving relatively low affinities (see Figure 24.19).

The respiratory-pigment physiology of individuals undergoes acclimation and acclimatization

When individual animals are exposed chronically to reduced O2 availability in their environments, they often respond with chronic alterations of their respiratory-pigment physiology. The most com- mon response of this sort in both vertebrates and invertebrates is for the concentration of the respiratory pigment in the blood to be increased. Fish, for example, often increase the concentration of red blood cells in their blood when they live in poorly oxygenated waters.

In addition to the “quantitative strategy” of increasing the amount of respiratory pigment per unit of blood volume, animals also often modify the O2-binding properties of the pigments. Sometimes this is achieved by synthesizing different molecular forms. A dramatic example is provided by the water flea, Daphnia, a small, hemoglobin-synthesizing crustacean common in freshwater ponds. When Daphnia that have been living in O2-rich water are transferred into O2-poor water, hypoxia-inducible transcription fac- tors (HIFs) are released and affect DNA transcription by modulating hypoxia-response elements in the promoter regions for the globin genes (see page 604). Multiple globin types can be synthesized, and after the transfer to O2-poor water, the mix of globins is modified. In fact, new mRNAs can appear within minutes, and new hemoglobin molecules—composed of different proportions of globin subunits than the preexisting molecules—can appear within 18 h. The new molecular forms of hemoglobin have a higher O2 affinity than the preexisting ones. Thus, over the first 11 days in O2-poor water, the O2 affinity rises (P50 falls) as the concentration of hemoglobin also rises (FiguRE 24.20)! Together, these changes give the Daphnia a greatly enhanced capability to acquire O2 from their environment. The Daphnia also, as mentioned earlier, change color. Pale at the start, they turn brilliant red (see Figure 24.20).

For modifying the O2-binding properties of respiratory pig- ments, perhaps a more common strategy is not to alter the molecular forms of pigments synthesized but to modulate preexisting types in advantageous ways. When fish, for example, are transferred from well-aerated to poorly aerated waters, they do not typically alter their hemoglobin types, but they often decrease the concentrations of ATP and GTP within their red blood cells over time. These chronic changes in the intracellular modulators of hemoglobin raise its O2 affinity. Blue crabs, as noted earlier, chronically raise the concentration of Ca2+ in their blood when exposed to O2-poor

20 Figure 24.16, although it applies to changes in O2 affinity within a species, illustrates this effect.

25

FiguRE 24.19 The O2 affinity of the hemoglobin in the whole blood of primates is a regular function of body size Small- bodied species tend to exhibit lower O2 affinity—and thus higher P50—than large-bodied ones. (After Dhindsa et al. 1972.)

of 2.1–2.4 kPa (16–18 mm Hg).19 One reason goldfish survive the tender loving care of kindergarteners is that these members of the carp family have high-affinity hemoglobins that can load well in O2-poor water. Mammal species that live underground typically have evolved higher O2 affinities than aboveground species of the same body size. Similarly, some species native to high altitudes have evolved higher O2 affinities than lowland species (see Box 24.5).

Another thought-provoking evolutionary pattern that has been

discovered is the relation between O2 affinity and body size in

groups of related species. In mammals and some other vertebrate

groups, the O2 affinity of blood hemoglobin tends to decrease as

body size decreases: Small species have relatively high P50 values

and therefore relatively low O2 affinities (FiguRE 24.19). Natural

selection is hypothesized to have favored this pattern because of

the inverse relation between weight-specific metabolic rate and

body size (see Chapter 7). Arterial blood oxygenates similarly in

all species of aboveground mammals near sea level because the

O2 partial pressure in the lungs is high enough in all such species

to be on the plateaus of their oxygen equilibrium curves (where

differences in affinity have little effect; see Figure 24.16). The

1 10
Body weight (kg) on log scale

lower-affinity hemoglobins in the smaller species unload O2 to

Hill Animal Physiology 4E
the tissues more readily, however. In this way, the lower affinity in

Sinauer Associates
the smalMl sopraelecsieSstuisdiohypothesized to help them meet their higher

Figure 24.19 12-11-15

weight-specific O2 needs.
Of course, it is exciting to find trends that make sense, but

sometimes when physiologists have compared the O2 affinities of related species, they have found no clear patterns, or even trends opposite to those expected. At present, a comprehensive predictive theory of affinity adaptation does not exist. An important reason is that when affinity is modified in the course of evolution, the changes can potentially affect both loading and unloading. Although a de- crease in affinity, for example, could aid O2 delivery by promoting O2

19 These measurements were made at approximately the same CO2 partial pressures and temperatures.

100 1000

3

Ring-tailed lemur

Black lemur Gibbon

Human Chimpanzee

Rhesus mon

key

Orangutan

Gorilla

P50

Hill Animal P Sinauer Assoc Morales Studio Figure 24.20

1.2

The O2 affinity
of hemoglobin dramatically rises 1.0 (P50 falls) because
of synthesis of
new molecular
forms while 0.8 simultaneously...

500 400

waters, apparently by mobilizing Ca2+ from the exoskeleton; the effect is to raise the O2 affinity of their hemocyanin. BOx 24.5 discusses how mammals respond to the low atmospheric O2 partial pressures of high altitudes.

Icefish live without hemoglobin

We now end our discussion of the transport of O2 by respiratory pigments by recalling a group of unusual and puzzling vertebrates, the Antarctic icefish: animals that, although reasonably large, have no O2 transport by blood hemoglobin because they have

...the total concen- tration of hemoglobin in the blood sharply increases to a new plateau.

200

100

0.6 0.4

0.2

300 no hemoglobin in their blood. Of all vertebrates, the icefish— which we discussed at length in Chapter 3—are the only ones that lack blood hemoglobin as adults. As stressed earlier, their habitat is undoubtedly critical in permitting them to live without blood hemoglobin. The Antarctic seas tend to be consistently well aerated, and the temperature of the water is typically frigid (near –1.9°C) year-round. Because of the low temperature, the solubility of O2 is relatively high, not only in the ambient water but also in the fish’s blood. Despite the advantages of high O2solubility, the blood oxygen-carrying capacities of icefish (about 0.7 mL O2/100 mL) are only about one-tenth as high as those of related red-blooded Antarctic fish (6–7 mL O2/100 mL). The icefish circulate their blood exceptionally rapidly, evidently to compensate for the fact that each unit of blood volume carries relatively little O2. They have evolved hearts that are dramati- cally larger than those of most fish of their body size; with each heartbeat, they therefore pump at least four to ten times more blood than is typical. In the microcirculatory beds of their tis- sues, they also have blood vessels that are of exceptionally large diameter. These large vessels allow rapid blood flow to occur with exceptionally low vascular resistance.

0.0 0 0 2 4 6 8 10 12

Days after transfer to low-O2 water

FIguRe 24.20 When water fleas are transferred to O2-poor water, their O2-transport system undergoes rapid acclima- tion because of altered gene expression The water fleas (Daphnia magna) had been living in well-aerated water and were transferred at time 0 to water in which the O2 partial pressure (3 kPa) was only 15% as high as in well-aerated water.The composition and concentration of their blood hemoglobin were then monitored for 11 days. The animals change color, as shown by the photographs. Sym- bols are means; error bars delimit ± 1 standard deviation. (After Paul et al. 2004; photos courtesy of Shinichi Tokishita.)

Transport of Oxygen and Carbon Dioxide in Body Fluids 657

P50

Hemoglobin concentration

Hemoglobin concentration (μM heme)

P50 (kPa)

Blood respiratory pigments typically become well oxygenated in the breathing organs, and when animals are at rest, the respiratory pigments typically release only a modest fraction of their O2 to the systemic tissues (25% in humans). During exercise, O2 delivery is enhanced by increases in both the extent of pigment unloading and the rate of blood flow.

Carbon Dioxide Transport

Carbon dioxide dissolves in blood as CO2 molecules, but usually only a small fraction of the carbon dioxide in blood is present in this chemi- cal form (about 5% in human arterial blood). Thus the first step in understanding carbon dioxide transport is to discuss the other chemi- cal forms in which carbon dioxide exists in blood. Because carbon dioxide can be present in multiple chemical forms, not just CO2, we must distinguish the material from its exact chemical forms. We do this by speaking of “carbon dioxide” when we refer to the sum total of the material in all its chemical forms and by specifying the chemical form (e.g., CO2) when we refer to a particular form.

When carbon dioxide dissolves in aqueous solutions, it un- dergoes a series of reactions. The first is hydration to form carbonic acid (H2CO3):

partial pressure regardless of how much CO2 it donates to the solution. From Chapter 22, we know that after the solution comes to equilibrium with the gas, the concentration of carbon dioxide in solution in the form of CO2 will be simply proportional to the CO2 partial pressure. Thus the amount of CO2 taken up in dissolved form by our liter of solution will depend simply on the principles of gas solubility. In contrast, the extent of bicarbonate formation is governed, not by the principles of solubility, but by the action of compounds that act as buffers of pH. In blood, these are the blood buffers. For our immediate purposes, the function of the blood buffers that deserves emphasis is that, under conditions when the concentration of H+ is being driven upward, they are able to restrain the rise in concentration by removing free H+ ions from solution (we’ll return to a fuller description of buffer function shortly). –

How do blood buffers determine the amount of HCO3 mation? A straightforward way to see the answer is to return to the analysis of the solution mentioned in the last paragraph and apply the principles of mass action (see page 50) to Equation 24.5. According to the principles of mass action, the following equation holds true at equilibrium:

The second is dissociation of the carbonic acid to yield bicarbonate (HCO3–) and a proton:

H CO ~ H+ + HCO − 233

(24.4)

Bicarbonate can then dissociate further to yield carbonate (CO32–) and an additional proton. This final dissociation, however, occurs to only a small extent in the body fluids of most animals. Moreover, although carbonic acid is an important intermediate compound, it never accumulates to more than very slight concentrations. For most purposes, therefore, the reaction of CO2 with water can be viewed simply as yielding HCO3– and protons:

CO2 + H2O ~ HCO3− + H+ (24.5)

Equation 24.5 emphasizes that carbon dioxide acts as an acid in aqueous systems because it reacts to produce H+; as mentioned earlier, it has been aptly termed a “gaseous acid.”

The extent of bicarbonate formation depends on blood buffers

Almost no bicarbonate is generated when CO2 is dissolved in dis- tilled water or a simple salt (NaCl) solution. However, bicarbonate is typically the dominant form in which carbon dioxide exists in the bloods of animals. How can we explain these two, seemingly contradictory, statements? The answer lies in the factors that affect bicarbonate formation, which we now examine.

Suppose that we bring a liter of an aqueous solution—initially devoid of carbon dioxide—into contact with a gas that acts as a source of CO2, and that this gas remains at a constant CO2

Transport of Oxygen and Carbon Dioxide in Body Fluids 659

HCO_H+
3 =K (24.6)

[CO2 ]
where the square brackets signify the concentrations of the various

for-

CO + H O ~ H CO 2223

(24.3)

chemical entities, and K is a constant. Because [CO2] is a constant at equilibrium in our solution at a given CO2 partial pressure, and because K is also a constant, Equation 24.6 reveals that the amount of HCO3– formed per unit volume of solution depends inversely on the H+ concentration. If [H+] is kept relatively low, [HCO3–] at equilibrium will be relatively high, meaning that a lot of HCO3– will be formed as the system approaches equilib- rium. However, if [H+] is allowed to rise to high levels, [HCO3–] at equilibrium will be low, meaning little HCO3– will be formed. When carbon dioxide enters our solution from the gas and un- dergoes the reaction in Equation 24.5, the degree to which the H+ made by the reaction is allowed to accumulate, driving [H+] up, is determined by the buffers in the solution. If the buffers are ineffective, the H+ produced by the reaction will simply ac- cumulate as free H+ in the solution; thus [H+] will rise rapidly to a high level, and the entire reaction will quickly reach an end point with little uptake of carbon dioxide and little formation of HCO3–. However, if the buffers are highly effective, so that most H+ is removed from solution as it is formed, [H+] will stay low, and a great deal of carbon dioxide will be able to undergo reac- tion, causing a large buildup of HCO3–.

Let’s now speak about buffers in more detail. Buffer reactions are represented by the general equation

HX ~ H+ + X− (24.7)

where X– is a chemical group or compound that can combine reversibly with H+. When H+ is added to a buffered solution, the buffer reaction is shifted to the left, removing some of the H+ from free solution (as already stressed). However, if H+ is extracted from a buffered solution, the reaction shifts to the right, releasing free H+ from compound HX. In brief, a buffer reaction acts to stabilize [H+]. Together, HX and X– are termed a buffer pair. According to

660 Chapter 24
the principles of mass action, the following equation describes a

buffer reaction at equilibrium:

H+ X–
[HX] = K′ (24.8)

where K′ is a constant that depends on the particular buffer reaction and the prevailing conditions, notably temperature. The negative of the common logarithm of K′ is symbolized pK′, just as the negative of the logarithm of [H+] is called pH. The effectiveness with which a particular buffer reaction (a particular buffer pair) is able to stabilize [H+] is greatest when half of the X– groups are combined with H+ and half are not; that is, the change in pH caused by the addition or removal of H+ is minimized when [HX] = [X–]. From Equation 24.8,

–+
it is clear that for [HX] and [X ] to be equal, [H ] must equal K′; that

is, pH must equal pK′. Therefore the buffering effectiveness of any given buffer reaction is greatest when the prevailing pH matches the pK′ of the reaction. Applying this principle to the blood of an animal (it also applies to other solutions), we can say that the blood may contain an enormous variety of potential buffer pairs, but typi- cally the buffer reactions that are important will be those with pK′ values within one pH unit of the pH prevailing in the blood.

The blood of mammals and most other vertebrates is highly effective in buffering the H+ generated from CO2 because the blood has a high concentration of effective buffer groups. These groups are found mostly on blood protein molecules, especially hemoglobin! Two types of chemical groups are particularly noteworthy as buffer groups because they are abundant and have appropriate pK′ values: the terminal amino groups of protein chains and the imidazole groups found wherever the amino acid histidine occurs in protein structure. The imidazole groups are the dominant buffering groups. The buffering of human blood is so effective that when CO2 undergoes the reaction in Equation 24.5, forming HCO3– and H+, the buffer groups remove more than 99.999% of the H+ produced from free solution! This buffering permits a great deal of HCO3– to be formed. Thus the blood can take up a great deal of carbon dioxide.

Carbon dioxide transport is interpreted by use of carbon dioxide equilibrium curves

Blood equilibrium curves for carbon dioxide have interpretive value

similar to that of oxygen equilibrium curves. To understand the

use of carbon dioxide equilibrium curves, we must first establish

the meaning of the total carbon dioxide concentration of the

blood. Suppose that some blood is brought to equilibrium with an

atmosphere containing no CO2, so that the CO2 partial pressure

of the blood is zero. Suppose that the blood is then exposed to an

atmosphere containing CO2 at some fixed, positive partial pres-

sure. And suppose that as the blood comes to equilibrium with the

new atmosphere, we measure the total quantity of CO2 it takes up,

regardless of the chemical form assumed by the CO2 in the blood.

This quantity—the total amount of CO2 that must enter each unit

of blood volume to raise the blood CO2 partial pressure from zero

to any particular positive CO2 partial pressure—is termed the

blood’s total carbon dioxide concentration at that partial pressure.

A plot of the total carbon dioxide concentration as a function of

(A) Human arterial blood

70 60 50

Bicarbonate formation 40 in the blood accounts

for most of the
30 difference between

the total carbon

dioxide concentration 20 and the dissolved CO2

CO partial pressure is known as a carbon dioxide equilibrium 2

ana (sometimes called Lithobates catesbeianus); lobster, Panulirus Morales Studio

curve or carbon dioxide dissociation curve (FiguRE 24.21A). What determines the shape of the carbon dioxide equilibrium curve? In mammals, carbon dioxide exists in blood in three principal

vulgaris; lungfish, Neoceratodus forsteri; mackerel, Scomber scom- Figure 24.21 12-11-15

10 0

concentration.

0 10 20 30 40 50 60 70 80 mm Hg

0 4 8 kPa Partial pressure of CO2 in blood

(B) Blood of nine species

90

80

70

60

50

40

30

20

10

0 0 10 20 30 40 50 60 70 80 mm Hg

0 4 8 kPa Partial pressure of CO2 in blood

FiguRE 24.21 Carbon dioxide equilibrium curves (A) The carbon dioxide equilibrium curve of fully oxygenated human blood at normal body temperature.The portion of the total carbon dioxide concentration attributable to dissolved CO2 is shown at the bottom. (B) Carbon dioxide equilibrium curves for oxygenated blood of nine species at 15°–25°C. Because all curves were not determined at ex- actly the same temperature, some of the differences among curves maHyillariAsneimfroalmPhteysmioploegrya4tuEreeffects.Species:bullfrog,Ranacatesbei-

Sinauer Associates

brus; mudpuppy, Necturus maculosus; octopus, Octopus macropus; toadfish, Opsanus tau; turtle, Pseudemys floridana. (After Hill and Wyse 1989.)

Carbon dioxide equilibrium curve (total carbon dioxide concentration)

Dissolved CO2

Total carbon dioxide concentration (mL CO2/100 mL) Carbon dioxide concentration (mL CO2/100 mL)

chemical forms, and thus the total carbon dioxide concentration has three components. Two, as we have already discussed, are dis- solved CO2 and HCO3–. The third is carbon dioxide that is directly chemically combined (in a reversible manner) with amino groups on hemoglobin and other blood proteins, forming carbamate groups (—NH—COO–) (also called carbamino groups). Typically, in both mammals and other types of animals, the great prepon- derance of blood carbon dioxide is in the form of HCO3–; 90% of the carbon dioxide in human blood, for example, is in that form. The shapes of the carbon dioxide equilibrium curves of animals are thus determined largely by the kinetics of HCO3– formation in their bloods. This means that the shapes depend on the blood buffer systems: the concentrations of buffer groups, their pK′ values, and the extent to which their capacities for H+ uptake are being called upon to buffer acids other than CO2.

A diversity of carbon dioxide equilibrium curves is found in the animal kingdom (FiguRE 24.21B). If we compare air-breathing and water-breathing animals, we find that they typically operate on substantially different parts of their carbon dioxide equilibrium curves. The reason, as discussed in Chapter 23 (see Box 23.1), is that air breathers typically have far higher arterial CO2 partial pressures than water breathers do. For example, the systemic arterial CO2 partial pressure in resting mammals and birds breathing atmospheric air— being at least 3.3 kPa (25 mm Hg)—is far higher than that commonly observed in gill-breathing fish in well-aerated waters, 0.1–0.4 kPa (1–3 mm Hg). In air breathers, the CO2 partial pressure of blood rises from a high arterial value to a still higher venous value as the blood circulates through the systemic tissues, meaning that the part of the carbon dioxide equilibrium curve that is used is the part at relatively high CO2 partial pressures. In water breathers, by contrast, both the arterial and venous CO2 partial pressures are relatively low; the part of the equilibrium curve that is used by water breathers is therefore the steep part at relatively low CO2 partial pressures.

The Haldane effect:
The carbon dioxide equilibrium curve depends on blood oxygenation

The carbon dioxide equilibrium curve of an animal’s blood commonly changes with the state of oxygenation of the respiratory pigment (the O2-transport pigment) in the blood, a phenomenon named the Haldane effect after one of its discoverers. When a Haldane effect is present, deoxygenation promotes CO2 uptake by the blood, whereas oxygenation promotes CO2 unloading. Thus the total carbon dioxide concentration at any given CO2 partial pressure is greater when the blood is deoxygenated than when it is oxygenated (FiguRE 24.22). The reason for the Haldane effect is that the buffering function of the respiratory pigments—which play major buffer roles—depends on their degree of oxygenation. Deoxygenation of a respiratory pig- ment alters its buffering function in such a way that it tends to take up more H+ and lower the blood concentration of H+. According to Equation 24.6, this means that when a respiratory pigment becomes deoxygenated, more HCO3– can form, and the blood therefore reaches a higher total carbon dioxide concentration. This phenomenon is the necessary converse of the Bohr effect, as noted earlier (see page 648).

The functional significance of the Haldane effect is illustrated in the inset of Figure 24.22 using CO2 transport in resting humans as an example. Point A shows the total carbon dioxide concentration and CO2 partial pressure in arterial blood, whereas point V shows the values in venous blood. The arrows between A and V represent the functional relation between total carbon dioxide concentration and CO2 partial pressure in the body, where oxygenation changes simultaneously with the uptake and release of CO2. Note that the slope of this functional relation is steeper than the slope of any of the equilibrium curves in Figure 24.22 for blood at a fixed level of oxygenation (red and purple lines). Thus, when the CO2 partial pressure shifts back and forth between its values in arterial and venous blood (A and V), the blood takes up and releases more

70

60

50

40

30

20

10

00 10 20 30 40 50 60 70 mm Hg

0 4 8 kPa Partial pressure of CO2 in blood

FiguRE 24.22 The Haldane effect and its implications for human carbon dioxide transport The principal graph (left) shows carbon dioxide equilibrium curves for fully oxygenated and essentially fully deoxygenated human blood, illus- trating the Haldane effect.The inset (above) summarizes carbon dioxide transport in humans at rest. Point A represents arterial blood, which is fully oxygenated and has a CO2 partial pressure of about 5.3 kPa (40 mm Hg). Point V represents mixed venous blood, which is about 70% oxygenated and has a CO2 partial pressure

of about 6.1 kPa (46 mm Hg).The arrows show the functional relation between total carbon dioxide concentration and CO2 partial pressure as blood circulates through the body, becoming alternately arterial (A) and venous (V ).

Transport of Oxygen and Carbon Dioxide in Body Fluids 661

Total carbon dioxide concentration (mL CO2/100 mL)

60

50

Fully deoxygenated

V

A

70% oxygenated Fully oxygenated

50 60 mm Hg

40 30 40

4 6 8 kPa Partial pressure of CO2 in blood

Fully deoxygenated blood

Fully

oxygenated blood

Total carbon dioxide concentration (mL CO2/100 mL)

662 Chapter 24

CO2 than would be possible without the Haldane effect. In this way, hemoglobin function simultaneously aids CO2 transport and O2 transport!

Critical details of vertebrate CO
depend on carbonic anhydrase and anion transporters

An important attribute of the hydration of CO2 to form bicarbon- ate (Equation 24.5) is that it occurs relatively slowly in the absence of catalysis (requiring a minute or so to reach equilibrium). The native slowness of this reaction presents a potential bottleneck in the blood’s ability to take up CO2 as bicarbonate in the systemic tissues and release CO2 from bicarbonate in the lungs. The enzyme carbonic anhydrase (CA) greatly accelerates the interconversion of CO2 and HCO3–, thereby preventing this reaction from acting as a bottleneck.21 The reaction is the only one in CO2 transport known to be catalyzed.

The morphological location of CA has important consequences for CO2 transport. In vertebrates, CA is found within the red blood cells but almost never free in the blood plasma. Sometimes CA is

21 When CO is hydrated to form HCO – by carbonic anhydrase catalysis, 23

also found associated with the inner endothelial walls of blood capillaries, such as lung or skeletal muscle capillaries. A key point is that CA is both essential and localized.

With this in mind, let’s discuss what happens when CO2 from metabolism enters the blood in a systemic capillary (FIguRe 24.23). CO2 diffuses readily into the red blood cells (possibly mediated in part by aquaporin AQP-1). There it encounters CA and is quickly converted to HCO3– and H+. In fact, if there is no membrane-bound CA in the capillary walls or other CA outside the red blood cells, virtually all the reaction of CO2 to form HCO3– and H+ occurs inside the cells. Hemoglobin—the most important blood buffer—is immediately available inside the red blood cells to take up H+ and thus play its critical role in promot- ing HCO3– formation. In fact, because hemoglobin is undergoing deoxygenation as CO2 is added to the blood, hemoglobin develops a greater affinity for H+ just as it is needed. The red blood cell membranes of nearly all vertebrates are well endowed with a transporter protein—a rapid anion exchange protein (often termed the band 3 protein)—that facilitates diffusion of HCO3– and Cl– across the membranes in a 1:1 ratio. The HCO3– that is formed and buffered inside red cells thus tends to diffuse out into the

plasma, so that the plasma ultimately carries most of the HCO –

added to the blood in the systemic capillaries. As HCO3

H2CO3 is not formed as an intermediate. Instead, the reaction proceeds by a pathway not involving H2CO3 formation.

–3 diffuses

2

transport

Wall of blood capillary Cell membrane (endothelium)

Imidazole groups on hemoglobin (Hb) take up H+ in their role as buffers. They increase their affinity for H+ when Hb is deoxygenated.

Red blood cell

Some bicarbonate buffered in the RBC
is carried inside the RBC...

HCO3–

Cl–

Rapid anion exchange protein

HCO3–

H+ + HCO3–

...but most diffuses out of the RBC via the rapid anion exchange protein. This bicarbonate is carried in the plasma, although buffered in the RBC.

Pr– HPr

Bicarbonate produced in the plasma is buffered by plasma proteins (Pr).

+ + H

tion.The processes shown in this figure occur in reverse in the lungs or gills. Where the labels refer to the site of buffering, they are specifying where the H+ generated during bicarbonate production is taken up by buffering compounds. Carbamate formation occurs to a significant extent in mammals, but not necessarily in other vertebrates. Hb, hemo- globin; RBC, red blood cell; Pr, plasma proteins.

Im HbO2 NH2

Catalyzed hydration

Cl–

Chloride shift

+ H+ Carbamate formation

Carbonic anhydrase in the RBC catalyzes this reaction.

O2

CO2

H2O

Blood plasma

H2O

– HCO3

ImH+ Hb

NHCOO–

O2 CO2 Tissue (e.g., muscle)

Catalyzed hydration

Some vertebrate tissues have a membrane-bound carbonic anhydrase.

CO2

FIguRe 24.23 The major processes of CO2 uptake by the blood in a systemic blood capillary of a vertebrate The molecules boxed in red and purple (HbO2 and Hb) represent oxygen- ated and deoxygenated hemoglobin,respectively.Three elements of molecular structure are highlighted in each hemoglobin molecule: the O2-binding site (symbolized Hb), an imidazole buffering group (Im), and an amino group (NH2) that can participate in carbamate forma-

out of the red blood cells into the plasma, Cl– diffuses into the 8.5 cells from the plasma—a process called the chloride shift. In
tissues that have a membrane-bound CA associated with the blood-capillary endothelium, such as the skeletal muscles of at

least certain vertebrates, some rapid formation of HCO3– and 8.0 H+ occurs in the plasma, where the HCO3– must be buffered
by plasma proteins. All these events occur in reverse when the
blood flows through the lungs or gills.

The operations of these kinetic details govern the exact ways 7.5 in which CO2 is transported under any set of conditions. For
example, from recent research, we know that the operations of
the kinetic details differ during exercise and rest—a difference

that may ultimately prove to be critical for a full understanding of 7.0 exercise physiology.

The neutral
pH (the pH
of pure water) varies with temperature. The pH values of animal bloods often vary in parallel.

Acid–Base Physiology

The pH of the body fluids cannot vary far from normal levels with- out serious functional consequences. In humans, for instance, the normal pH of arterial blood at 37°C is about 7.4, and a person will lie near death if his or her pH rises to just 7.7 or falls to 6.8! Abnormal H+ concentrations inflict their adverse effects to a large extent by influencing the function of proteins. As buffer groups on a pro- tein molecule take up or lose H+, the electrical charge of the whole molecule is rendered more positive or negative; beyond certain limits, these changes result in changes in molecular conformation or other properties that interfere with proper protein function. For the electrical-charge and ionization status of a protein molecule

of humans and other large mammals gave rise to the notion that the pH in any particular region of the body is always regulated at a single, invariant level (e.g., 7.4 in human arterial blood). We now realize that this type of pH regulation is a special case that occurs only in animals that maintain a constant deep-body temperature. Considering all animals taken together, the pH that is maintained by acid–base regulatory mechanisms—whether in the blood or inside cells—is more commonly a temperature- dependent variable.

What advantage might animals gain by increasing their pH as their body temperature falls? According to the leading hypothesis, the alphastat hypothesis, the changes in pH are a means of main- taining a constant state of electrical charge on protein molecules.

Transport of Oxygen and Carbon Dioxide in Body Fluids 663

6.50 5 10 15 20 25 30 35 40 Body temperature (°C)

FiguRE 24.24 normal blood pH is a temperature-dependent variable As the neutral pH varies with body temperature, blood pH—which is more alkaline than neutral—often varies in parallel; in species that follow this pattern, the blood pH tends to remain alkaline to a fixed extent. Data are shown for six poikilothermic species and for humans. Species differ in how alkaline their blood is relative to the neutral pH. (After Dejours 1981.)

to remain within limits compatible with protein function, the pH must remain within parallel limits.

The neutral pH is defined to be the pH of pure water. By this definition, as shown in FiguRE 24.24, the neutral pH varies with temperature, being higher at low temperatures than at high ones. In poikilotherms, the normal blood pH often varies with body temperature in parallel with the neutral pH. Specifically, a com- mon pattern is that, within a species, the blood pH is displaced by a relatively fixed amount to the alkaline side of the neutral pH, rising and falling with body temperature to maintain this fixed displacement (see Figure 24.24). A species that follows this pattern is said to maintain a constant relative alkalinity of blood pH. In a species of this sort, the pH inside cells, the intracellular pH, also

Hipll arAanlilmelasl PnheyustioralolgpyH4E(although intracellular pH and blood pH are Sinauer Associates

different from each other).

Morales Studio

In the early days of the study of acid–base physiology, studies

Figure 24.24 12-11-15

pH

664 Chapter 24

The reason that changes in pH are required is that changes in temperature alter the chemical behavior of the buffer groups on protein molecules. Most importantly, as temperature falls, the pK′ values of imidazole groups increase. This means that at reduced temperatures, imidazole groups increase their inherent tendency to combine with H+. If this change in chemical behavior were unop- posed, more of the imidazole groups on proteins would be combined with H+ at low temperatures than at high ones. Decreasing the H+ concentration (raising the pH) at low temperatures serves to oppose the heightened tendency of the imidazole groups to take up H+. Accordingly, it helps prevent the proportion of positively charged groups on proteins from changing.

Acid–base regulation involves excretion + or retention of chemical forms affecting H concentration

When a process occurs that tends to cause a protracted increase in the amount of acid in the body of an animal, maintenance of the animal’s temperature-dependent normal pH requires that other processes be set in motion that will either export acid from the body or increase the body’s content of base. Conversely, if a dis- turbance occurs that decreases body acid, acid–base regulation requires a compensating uptake of acid or export of base. There are two competing “worldviews” of acid–base regulation.22 In our brief overview here, we adopt the simpler of the two, which focuses on adjustments in CO2, H+, and HCO3–.

The concentration of CO2 in the body fluids of an animal can be raised or lowered to assist acid–base regulation. This is especially true in terrestrial animals, which (in contrast to aquatic ones) normally have relatively high blood partial pressures of CO2. Suppose that a person’s blood becomes too acidic. One possible compensatory response is for the person to increase lung ventilation, thereby lowering the CO2 partial pressure in the blood and other body fluids. Lowering the blood CO2 partial pressure will pull Equation 24.5 to the left and thus lower the blood H+ concentration. Slowing of lung ventilation, by contrast, can assist with acid–base regulation if the body fluids become too alkaline. The slowing of ventilation will promote accumulation of CO2 in the body fluids and cause Equation 24.5 to be shifted to the right, providing more H+.

Animals often have the ability to exchange H+ itself with the environment, and this ability also can be used for acid–base regulation. Because H+ is not a gas, it must be transported in liquid solution. In terrestrial animals, responsibility for the export of H+ from the body rests with the kidneys. Humans, for example, are routinely confronted with an excess of H+ from their diet, and they void the excess principally in their urine; this urinary elimina- tion of H+ can be curtailed entirely, however, when appropriate. In aquatic animals, including both fish and crustaceans, H+ is exchanged with the environment by the gill epithelium (see Box 5.2, for example).

Bicarbonate ions are also exchanged with the environment to assist acid–base regulation. The HCO3– exchanges are mediated

22 The books by Davenport and Stewart in the References and Additional Readings, respectively, provide readable introductions to these two worldviews. For those who become interested in the strong ion difference approach, not covered here, the reference by Johnson et al. in the Additional Readings is also worthwhile.

principally by the kidneys in terrestrial animals but, it appears, principally by the gill epithelium in fish and crabs (see Box 5.2). Bicarbonate functions as a base. If retention of HCO3– in the body is increased, Equation 24.5 is shifted to the left, tending to remove H+ from solution in the body fluids, making the body fluids more alkaline. Conversely, increased elimination of HCO3– tends to raise the H+ concentration of the body fluids.23

Disturbances of acid–base regulation fall into respiratory and metabolic categories

Disturbances of the pH of the body fluids are categorized as acidosis or alkalosis. Acidosis occurs when the pH of the body fluids is shifted to the acid side of an animal’s normal pH at a given body temperature. Alkalosis is a shift in pH to the alkaline side of an animal’s normal pH. Disturbances of pH are also classified as respiratory or metabolic according to their primary cause.

The respiratory disturbances of pH are ones that are brought about by an abnormal rate of CO2 elimination by the lungs or gills. Respiratory alkalosis arises when the exhalation of CO2 is abnormally increased relative to CO2 production, causing the CO2 partial pressure in the body fluids to be driven below the level needed to maintain a normal pH. Panting by mammals, for example, sometimes causes respiratory alkalosis (see page 272). Respiratory acidosis occurs when the exhalation of CO2 is impaired and metabolically produced CO2 therefore accumulates excessively in the body. Prolonged breath-holding, for example, can cause respiratory acidosis.

Whereas the blood property that is initially altered in respiratory disturbances of pH is the CO2 partial pressure, metabolic distur- bances of pH—by definition—initially alter the blood bicarbonate concentration. metabolic alkalosis and metabolic acidosis both have numerous possible causes. Metabolic acidosis, for example, can result from excessive loss of HCO3– in gastrointestinal fluids during chronic diarrhea. Metabolic acidosis can also result from excessive addition of H+ to the body fluids, as when lactic acid is accumulated during vigorous exercise; the added H+ from lactic acid reacts with the pool of HCO3– in the body fluids, lowering the concentration of HCO3–.

Animals typically respond to disturbances of pH by marshal- ing their acid–base regulatory mechanisms. Lung ventilation by human athletes performing work of ever-increasing intensity provides a striking and interesting example. When athletes are not accumulating lactic acid, they simply increase their rate of lung ventilation in parallel with their rate of CO2 production. However, when athletes work intensely enough that they accumulate lactic acid, they increase their rate of lung ventilation more than their rate of CO2 production. This disproportional increase in ventilation, an example of hyperventilation, causes CO2 to be exhaled from the body faster than it is being produced. The CO2 partial pressure in the blood and body fluids is thereby lowered, helping to limit the degree of acidosis caused by the accumulation of lactic acid.

In the study of global climate change, a concern that has recently become a primary focus is ocean acidification (BOx 24.6).

23 One way to view this effect of HCO3– elimination is to recognize that HCO3– originates from H2CO3; when HCO3– is eliminated, just the H+ of H2CO3 remains in the body fluids, acidifying them.

Transport of Oxygen and Carbon Dioxide in Body Fluids 665 BOx Acidification of Aquatic Habitats

24.6

Compared with the bloods of animals, the waters of most natu- ral aquatic habitats, such as the oceans, are not buffered in a way that would significantly impede a rise in H+ concentration caused by addition of acidic materials. Acid rain has been a recognized problem in bodies of freshwater—streams, ponds, and lakes—for many decades. It is often caused by sulfur and nitrogen oxides liberated into the atmosphere by the combus- tion of fossil fuels.The oxides react with water to form acids, such as sulfuric acid.

Acidification caused by atmospheric CO2 is a more recently identified challenge.The CO2 concentration of Earth’s atmo- sphere has risen from about 300 parts per million (ppm) to about 400 ppm in the past century because of the burning

of wood, coal, and petroleum. Because of the principles we have discussed, the CO2 concentration in the waters of the ocean has increased (see Chapter 22), and this has made the oceans more acidic by about 0.1 pH unit by driving the reac- tion shown in Equation 24.5 to the right. Animals are not always able to regulate processes that are affected by the environ- mental acidification. For example, the acidification tends to erode certain types of animal skeletons that are composed of calcium carbonate (e.g., skeletons of reef corals) or to inter- fere with production of such skeletons. It can also interfere with sensory and developmental processes. Box Extension 24.6 provides references for further reading.

Rising atmospheric CO2 acidifies oceans, leading to chemical reactions that can dissolve calcium carbonate skeletons

Summary

Acid–Base Physiology

The neutral pH varies with temperature, being higher
at low temperatures than at high ones. In animals with variable body temperatures, the normal blood pH often varies in parallel with the neutral pH, being displaced in the alkaline direction to a constant extent (constant relative alkalinity).

Acidosis and alkalosis are categories of acid–base disturbance. They occur, respectively, when the blood pH is to the acid or alkaline side of an animal’s normal pH for the prevailing body temperature. Either sort of disturbance can be respiratory (originating because of changes in CO2 loss by breathing) or metabolic (originating because of changes in the blood bicarbonate concentration).

Within their range of acid–base regulation, animals correct chronic acid–base disturbances by modulating the elimination of CO2, H+, and HCO3– in regulatory ways