clinical chem 2

building blocks of proteins

influence:

\-cellular growth

\-cellular repair

\-cell maintenance

\-protein biological activity

basic structure of protein

\-amino group

\-carboxyl group

\-differ by R group

zwitterion

zero net charge

at physiological pH 7.4

carboxyl gives a hydrogen atom; amino acid receives

at low pH

positive

at high pH

negative

high water solubility

polar/charged

low water solubility

aliphatic/aromatic

peptide bond

Amino group (1st amino acid) linked with carboxyl group (2nd amino acid)

polypeptide

Chain of amino acids linked by peptide bonds

\

protein

large polypeptide

metabolism has two forms of amino acids

essential: nutrient dependant

nonessential: naturally occuring

proteolytic enzymes

Digest dietary proteins into constituent amino acids

absorption

Rapidly absorbed from intestine into bloodstream

functions of essential and non-essential amino acids

\-synthesis of body proteins

\-synthesis of non-protein nitrogen containing compounds

\-energy source

Aminoacidopathies

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\

Class of inherited errors of metabolism \n • Enzyme defect inhibits the body’s ability to metabolize certain amino acids

Phenylketonuria (PKU)

\

Absence of enzyme that converts phenylalanine to tyrosine

absence of enzyme needed to create cofactor for PAH (have it but can’t make it work)

Phenylketonuria (PKU)

symptoms and testing

Cause urine to have characteristic “musty” or “mousy” odor

Required test on newborn screening

Tyrosinemia

Type 1,2,&3

\-type 1 most severe

\-all enzyme mutations

Tyrosinemia diagnosis and treatment

increased levels of tyrosine

Low-protein diet \n Medication

Alkaptonuria

\

ack of enzyme homogentisate oxidase

Turns urine brown/black in presence of oxygen

deposits in cartilage of ears, nose, and tendons of \n extremities \n • Dark blue-black pigmentation (ochronosis)

Alkaptonuria test and treatment

Test: Ferric chloride test \n Treatment: Vitamin C supplements

Maple Syrup Urine Disease (MSUD) characteristics and testing

\

Patient’s urine, breath, and skin smell like burnt sugar

Testing: \n • Modified Guthrie Test \n • Microfluorometric assay

Homocystinuria

\

gene mutation affecting vision

Citrullinemia

Belongs to a class of genetic diseases called “Urea Cycle Disorders”

type 1: lack enzyme, accumulate ammonia in urine

type 2: mutation in gene for transport protein

Cystinuria characteristics and test

mutation for protein responsible for reabsorption of cystine from urine

color change

protein sample types

\-blood samples after 6-8 hour fast

\-heparin tube

\-non-hemolyzed

\-deproteinized

\-immediate testing or frozen

\-urine is random on 24hr specimen

Amino Acid Screening

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Thin layer chromatography (dyed blue)

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Amino Acid - Properties charge of side chain influenced by

• pH of solution \n • pKa of the side chain \n • Side chain’s environment

isoelectric point

pH where the net charge is zero

at acidic ph

cationic form

at alkaline ph

anionic form

carboxyl group

pK1

amino group

pK2

pK

pH at which equal quantities

Dissociation Constants

\

energy for large molecule to break down

amino acids can act as

buffers in two pH regimes

purpose of Calculate pH of Amino Acid w/Ionizable \n Side Chain

Identify species that carries a net zero \n charge

arginine (Arg)

conversion of ammonia into urea

histidine (his)

precursor to histamine

isoleucine (ilu), leucine (leu), valine (val)

branched chain amino acid

isoleucine

hemoglobin formation

leucine

optimal infant growth

valine

treatment for muscle, mental, and emotional problems

lysine (lys)

basic pH

function:

-antibody production

-triglyceride lower

methionine (met)

innate translation of mRNA

phenylalanine (phe)

promote alertness, mood elevation

threonine (thr)

collagen, elastin, and tooth enamel

tryptophan (trp)

precursor for serotonin,melatonin, and niacin

relaxant

alanine (ala)

-transfer of nitrogens from peripheral tissues to liver

-production of antibodies

-reduction of toxic substance build up

asparagine (asn)

-transport of nitrogen

-conversion of one amino acid into another

aspartic acid (asp)

-precursor for other amino acids

-generation of glucose from non sugar substrates

cysteine (cys)

not to be confused with cystine

-potentially toxic

-structural and functional component of many proteins and enzymes

glutamic acid (glu)

-Glutamate

-net negative charge by pH (polar)

-neurotransmitter

glutamine (gln)

-most abundant in the body

-transport ammonia to liver

proline (pro)

-strengthening of joints,, tendons, and cardiac tissue

-works with vitamin C to promote healthy connective tissue

glycine (gly)

-synthesis of numerous important compounds in the body

-slow muscle degeneration

-improve glycogen storage

serine (ser)

-metabolism of lipids and fatty acids

-protect myelin sheaths

tyrosine (tyr)

-precursor of adrenal hormones (epinephrine, norepinephrine, and dopamine)

protein simple functions

-energy

-colloidal osmotic pressure

-buffers

-protection

-hemostasis

protein functions advanced

-catalyze biochemical reactions (enzymes) for glycolytic pathway and dna replications/polymerase

-transporter (hemoglobin, lactose permease)

-receptors for hormones

-cell structure and support (collagen and keratin)

-immunity (antibodies)

-motion (myosin and actin)

-storage (ferritin)

protein consists of

carbon, oxygen, hydrogen, nitrogen*, and sulfer

polymers made from one or more unbranched chains of amino acids (peptide bonds)- some are small some are big

peptides

small condensation of amino acids

small in comparison to proteins

primary

simplest structure cannot be broken down

tertiary amino acid

secondary amino acid

quaternary amino acid

denaturation

break down and destruction of molecular aspects

protein synthesis done in the

liver

antibodies made in

plasma cells

protein synthesis is equal to

protein catabolism (protein build up and break down)

diurnal rhythm

increased during day (when eating)

decreased during night (when not eating)

hormones that control protein synthesis

-thyroxine

-growth hormone

-insulin

-testosterone

protein digested in

digestive tract, kidneys, and liver

protein digestions process

hydrolyzed amino acids

nitrogen

nitrogen balance

(N Usage)/(N Intake) = Excretion

negative balance

(N Usage)/(N Intake) < Excretion

positive balance

(N Usage)/(N Intake) > Excretion

protein break down results into

ammonia and ketoacids

protein classification

-simple or conjugated

-function

-composition

-migration in electrical field (electrophoresis)

-centrifuge (density)

-size

-location

simple proteins

yield amino acids

conjugated proteins

simple proteins combined with some nonprotein material in body

apo protein

forms a particular biochemical molecule such as a hormone or enzyme

amphoteric

either acid or base

pH>pi

protein will be negatively charged

pH<pi

protein will be positively charged

proteins least soluble at

isoelectric point

protein is found in all body fluids but the lab is focused on

-serum

-urine

-csf

plasma proteins

most frequently analyzed of all proteins

total protein equation

total protein= albumin + globulins

total nitrogen

measures all chemically bound nitrogen in specimen performed on various sample types

total protein concentration on reference range

concentration lower at birth

reference interval: 6.5-8.3 g/dL

kjeldahl method

sample is digested with acid to convert nitrogen in the protein to ammonium ion

-very accurate and precise

-but time consuming, inconvenient, and impractical for routine use

biuret method

widely used and easily automated

-cupric ions (Cu+2) bind to components in peptide bonds

-in presence of an alkaline medium, and minimum of 2 peptide bonds, a color change from blue to violet occurs

-can be affected slightly by bilirubin or lipemia, and the presence of proline

in biuret method absorbance is is proportional to what bonds

peptides

dye-binding method

-based on the ability of most serum proteins to bind to dyes

-dyes are used to stain the proteins after electrophoresis

-calibration can be difficult to define

-good sensitivity and low interference from small compounds

dye-binding method : coomassie brilliant blue 250 shifts the absorbance maximum of the dye from 465→

595 nm

refractometry

technique that measures how light is refracted when it passes through a given substance

-simple, quick, requires small volume

-not as accurate for nitrogen proteins

refractometer protein range

<3.5 g/dL