MSMS Exam II (Semester II)

Gross anatomy, embryology, and imaging; biochemistry I; physiology I

How does 2,3-biphosphoglycerate (2,3-BPG) alter the structure of hemoglobin?

2,3-BPG binds to the positive central cavity of deoxygenated hemoglobin, thus stabilizing the T-state and lowering hemoglobin's affinity for oxygen.

What structural difference exists between HbA and HbF (fetal hemoglobin) that accounts for HbF having a significantly lower p50 value?

HbF has no B chains, but has Y chains which lack the positively charged Histidine 143 in its central cavity to which 2,3-BPG can bind.

What is the biochemical basis for sickle cell anemia?

A hydrophilic glutamic acid gets replaced with a hydrophobic valine at the 6th amino acid position. This creates a "sticky patch", so when oxygen levels are low and the hemoglobin is in T-state, the hydrophobic patch slots into a complementary pocket on the next hemoglobin molecule.

What is an oxidoreductase?

An enzyme that catalyzes oxidation-reduction reactions (electron transfers), often using NAD+/NADH or FAD/FADH2 focators.

What type of enzyme is lactate dehydrogenase?

An oxidoreductase.

What is a transferase?

An enzyme that transfers functional groups between molecules.

What enzyme class is lactase? A protease?

Lactases and proteases are hydrolases.

What is a lysase?

An enzyme that cleaves C-C, C-O, and C-N bonds by means other than hydrolysis or oxidation that leaves behind double bonds or rings.

What enzyme class is aldolase? Decarboxylase?

Aldolases and decarboxylases are lysases.

What is an isomerase?

An enzyme that catalyzes the interconversion of isomers.

A mutase is an example of what type of enzyme?

An isomerase.

What is a ligase?

Ligases are enzymes that join two molecules together coupled with ATP cleavage.

What type of enzyme if pyruvate carboxylase?

A ligase.

What type of enzyme is a synthetase?

A ligase.

ATP-dependent kinases require what inorganic cofactor?

Mg2+

Carbonic anhydrase requires what inorganic cofactor?

Zn2+

What two groups comprise organic coenzymes?

Cosubstrates transiently bind and dissociate.

Prosthetic groups are covalently/tightly bound helper groups.

Provide two examples of prosthetic groups.

Biotin on carboxylases, FAD.

What is the difference between a kinase, a phosphorylase, and a phosphatase?

Kinase uses ATP to move a phosphate group.

Phosphorylase uses inorganic phosphate to cleave a bond and add a phosphate.

Phosphatase uses water to remove a phosphate group.

Kinase and phosphorylase are transferases, but phosphatase is a hydrolase.

What is the structure of myoglobin?

A monomer with 1 polypeptide chain and 1 heme group.

What is the structure of hemoglobin?

A tetramer, where each subunit contains a heme group (4 total). HbA is a2B2.

What is a right shift in the oxygen-hemoglobin dissociation curve?

A decreased affinity / increased p50. The hemoglobin delivers oxygen.

What factors cause a right shift in the oxygen-hemoglobin dissociation curve?

CADET, face right!

(Increased CO2, increased acidity, increased 2,3-DPG, exercise, temperature)

Alternatively mnemonic:

BAT CAVE

(2,3-BPG, acid, temperature, CO2, altitude, vigorous exercise)

Describe the Bohr effect.

Hard-working tissues like exercising muscles create lots of CO2 and H+ ions (acid), lowering the blood's pH. The H+ and CO2 build salt bridges to stabilize the T state and attach to hemoglobin, decreasing its affinity for oxygen and causing oxygen release where the cells need it most.

Low pH is…

Acidic.

High pH is…

Basic.

Increased p50 confers…

Decreased affinity for oxygen.

Decreased p50 confers…

Increased affinity for oxygen.

What is the point mutation present in sickle cell anemia (HbS)?

A negatively charged glutamic acid is replaced with a nonpolar valine.

Describe methemoglobinemia.

Fe2+ (ferrous) is oxidized to Fe3+ (ferric), which cannot bind oxygen. The remaining Fe2+ is forced into the high-affinity R-state that prevents oxygen unloading.

Clinically presents as cyanosis with chocolate-brown blood. Treated with methylene blue.

What is the biochemical basis of carbon monoxide poisoning?

CO has a 200x greater affinity for hemoglobin than oxygen. It locks hemoglobin subunits in the high-affinity R-state, thus shifting the curve left and preventing oxygen release.

When hyperphosphorylated, the Tau protein is implicated in…

Alzheimer's disease. Typically, it helps stabilize microtubules.

The SIRT1 protein is implicated in…

Cardiovascular disease.

How does SDS-PAGE work?

The native charges of proteins are masked, so they are separated based exclusively on absolute size. The anionic detergent destroys the natural shapes so all proteins become linear.

Cannot purify proteins, only analyze.

IEF (isoeleectric focusing) works by…

Separating proteins along a gel with a pH gradient under an electric field pased on their isoelectric point. When the protein enters a gel zone where the local pH equals its pI, its net charge becomes zero and it stops moving.

Describe western blots.

Proteins are separated by SDS-PAGE, then incubated with a primary antibody specific to a target protein, then a second antibody conjugated to horseradish peroxidase (HRP) for signal detection.

Provides semi-quantitative comparisons of relative protein expression levels between samples.

Describe ELISA.

Capture antibodies are immobilized in microtiter wells help to bind target proteins, and then a detection antibody system is used.

High throughput quantitative analysis of soluble proteins, like hCG in pregnancy tests!

Describe IHC (immunohistochemistry).

Primary and secondary antibodies are applied to tissue sections to generate a localized DAB precipitate under microscopy. This preserves cellular architecture to determine which cells within a tissue biopsy express a biomarker.

HER2/neu is a biomarker implicated in…

Breast cancer.

SMAD4 is a biomarker implicated in…

Pancreatic islet/cancer cells.

Name two electrophoresis methods gone over in class materials.

SDS-PAGE (which separates proteins based on absolute size) and IEF (that separates proteins based on charge).

What is X-Ray Crystallography?

Determines the 3D protein structure of a crystalized protein with high resolution.

What is NMR Spectroscopy?

Nuclear Magnetic Resonance spectroscopy analyzes proteins in a liquid state since it is closer to physiologic conditions.

What is Co-IP?

Co-immunoprecipitation uses antibodies against Protein A to capture it from a lysate. If Protein B binds to Protein A, then Protein B will precipitate and be seen on a Western Blot.

What is the value of Co-IP?

It allows a scientist to verify the physical interactions between proteins.

By capturing BAD with anti-BAD antibodies, BCL2 is precipitated. This confirms that BAD and BCL2 form a physical complex. To discover this, a scientist would use…

Co-immunoprecipitation (Co-IP)

When performing an SDS-PAGE gel electrophoresis, what is the function of the SDS?

The SDS coats the proteins with a uniform negative charge so the separation is based exclusively off of molecular size.

A scientist is performing IEF (isoelectric focusing). A protein moves until a certain point, then stops. Why did it stop moving?

Because the net charge = 0. The gel's pH at the spot it stopped equals the protein's pI.

What is a lipophilic messenger?

Lipophilic messengers traverse lipid plasma membranes directly to bind to a receptor. The ligand-receptor complex then acts as a transcription factor and binds Hormone Response Elements (HREs) to alter gene elements in delayed, long-term response.

What is a hydrophilic messenger?

Hydrophilic messengers can't pass through cell membranes like lipophilic messengers can. Instead, they bind to cell-surface receptors like GPCRs, RTKs, and ligand-gated ion channels to initiate rapid second-messenger cascades.

What is the typical total blood volume of a regular-sized male? What percentage of it is in systemic circulation at one time? Pulmonary circulation? In the cardiac chambers?

Approximately 5 L.

84% is in systemic circulation

9% is in pulmonary circulation

7% is in the cardiac chambers at the end of diastole.

Why are veins called capacitance vessels?

Because 75% of the body's total blood volume exists in the veins, and veins easily stretch to accommodate it. Only 25% remains in arteries and capillaries.

Where in the body's circulation is pressure highest?

Pressure is highest in the aorta and large arteries.

Where in the body's circulation is pressure lowest?

Pressure is lowest in the vena cava.

Where in the body does pressure of blood significantly drop?

Pressure drops sharply in the arterioles, which are the site of the highest resistance given their high-percentage makeup of smooth muscle.

Describe the pathway of blood.

Aorta, artery, arteriole, vein, capillary.

All animals appreciate veiny… chickens.

What is the composition of a capillary?

Only a thin layer of epithelium to allow easy exchange of gas.

Describe the equation to find pulse pressure.

Pulse pressure = systolic pressure - diastolic pressure

Increasing stroke volume and decreasing arterial compliance will do what to pulse pressure?

Increase it.

As an individual ages, the elastin of the large arteries is replaced by collagen. This reduces arterial compliance, which does what to pulse pressure?

Increases pulse pressure.

What is mean arterial pressure?

The average pressure over a complete cardiac cycle.

Describe mean arterial pressure mathematically. Explain why it is not a perfect average.

MAP = P diastole + 1/3(P systole - P diastole)

A greater portion of the cardiac cycle is spent in diastole than in diastole.

What is systole?

Contraction.

What is diastole?

Relaxation.

Increased total peripheral resistance will do what to mean arterial pressure?

MAP = CO * TPR

Increased TPR will increase the mean arterial pressure.

Mathematically describe cardiac output.

CO = HR * SV