Oxygen binding proteins

Which of the following is not correct concerning 2,3-bisphosphoglycerate (BPG)? A) It binds at a distance from the heme groups of hemoglobin. B) It binds with lower affinity to fetal hemoglobin than to adult hemoglobin. C) It increases the affinity of hemoglobin for oxygen. D) It is an allosteric modulator. E) It is normally found associated with the hemoglobin extracted from red blood cells.

C

Which of the following is not correct concerning cooperative binding of a ligand to a protein? A) It is usually a form of allosteric interaction. B) It is usually associated with proteins with multiple subunits. C) It rarely occurs in enzymes. D) It results in a nonlinear Hill Plot. E) It results in a sigmoidal binding curve.

C

Which is stronger CO or O2 bound to hemoglobin?

CO, once bound O2 cannot displace it

________ is produced by metabolism in tissues

CO2

CO2 + H2O

H+ + HCO3-

actively metabolizing tissues generates

H+ ions

O2 is ________ to the _____ further facilitating the binding of O2

H-bonded distal His

hemoglobin consist of ___________ chains and _______ chains

2 alpha 2 beta

CO binds over _________ times better than O2

20,000

despite protein pocket CO still binds about ________ times better than O2

250

________ in plane and _______ perpendicular to it

4x 2x

normal concentration of BPG in normal human blood at sea level

5mM

iron atom of heme has _________ coordination bonds

6

how many helical segments in myoglobin?

8

concentration of BPG at high altitudes

8mM

In the binding of oxygen to myoglobin, the relationship between the concentration of oxygen and the fraction of binding sites occupied can best be described as: A) hyperbolic. B) linear with a negative slope. C) linear with a positive slope. D) random. E) sigmoidal.

A

The energy that is released by the hydrolysis of ATP by actin is used for: A) actin filament assembly. B) actin filament disassembly. C) actin-myosin assembly. D) actin-myosin disassembly. E) muscle contraction.

A

The proteins of the Major Histocompatibility Complex (MHC) bind and display: A) antigen fragments. B) B cell fragments. C) immunoglobin fragments. D) macrophage fragments. E) T cell fragments.

A

Which of the following generalizations concerning motor proteins is correct? A) They convert chemical energy into kinetic energy. B) They convert chemical energy into potential energy. C) They convert kinetic energy into chemical energy. D) They convert kinetic energy into rotational energy. E) They convert potential energy into chemical energy.

A

In hemoglobin, the transition from T state to R state (low to high affinity) is triggered by: A) Fe2+ binding. B) heme binding. C) oxygen binding. D) subunit association. E) subunit dissociation.

C

the 8 segments are labeled

A through H

Explain why most multicellular organisms use an iron-containing protein for oxygen binding rather than free Fe2+. Your answer should include an explanation of (a) the role of heme and (b) the role of the protein itself.

Ans: (a) Binding of free Fe2+ to oxygen would result in the formation of reactive oxygen species that can damage biological structures. Heme-bound iron is less reactive in this regard. (b) Binding of oxygen to free heme can result in irreversible oxidation of the Fe2+ to Fe3+ that does not bind oxygen. The environment of the heme group in proteins helps to prevent this from occurring.

a) What is the effect of pH on the binding of oxygen to hemoglobin (the Bohr Effect)? (b) Briefly describe the mechanism of this effect.

Ans: (a) The affinity decreases with decreasing pH. (b) At lower pH (i.e., higher H+ concentration) there is increasing protonation of protein residues such as histidine, which stabilizes the low affinity conformation of the protein subunits.

Describe briefly the basic structure of an IgG protein molecule.

Ans: An IgG protein contains two copies of a large polypeptide (heavy chain) and two copies of a small polypeptide (light chain). b structure contributes significantly to the tertiary structure of domains of both chains. Disulfide bonds link the heavy chains to one another and to the light chains. The chains are arranged in a Y-shaped structure where the two arms are linked to the base by a protease sensitive ("hinge") region.

Describe how you would determine the Ka (association constant) for a ligand and a protein.

Ans: An experiment would be carried out in which a fixed amount of the protein is incubated with varying amounts of ligand (long enough to reach equilibrium). The fraction of protein molecules that have a molecule of ligand bound is then determined. A plot of this fraction (q) vs. ligand concentration [L] should yield a hyperbola. The value of [L] when q = 0.5 is equal to 1/Ka.

Why is it likely that the immune system can produce a specific antibody that can recognize and bind to any specific chemical structure?

Ans: As a result of genetic recombination mechanisms, antibody-producing B cells are capable of producing millions of different antibodies with different binding specificities.

How does BPG binding to hemoglobin decrease its affinity for oxygen?

Ans: BPG binds to a cavity between the b subunits. It binds preferentially to molecules in the low-affinity T state, thereby stabilizing that conformation.

What is the concept of "induced fit" as it applies to antigen-antibody binding?

Ans: The conformations of the antigen and antigen-binding site of the antibody are influenced by each other and change as binding occurs. These conformational changes increase the chemical complementarity of the sites and result in tighter binding.

Explain how the effects of sickle cell disease demonstrate that hemoblobin undergoes a conformational change upon releasing oxygen.

Ans: In Hemoglobin S, the wild-type glutamate at residue 6 of the B-chain is replaced by valine. When oxygen is bound, both Hemoglobin A and Hemoglobin S are soluble, but in the deoxy- form. Hemoglobin S (but not Hemoglobin A) becomes very insoluble, due to exposure of the hydrophobic valine residue. This exposed "patch" causes aggregation of deoxy-Hemoglobin S into long insoluble fibrous aggregates, resulting in distorted shapes of the red blood cells (and leading to the symptoms of the disease). (See p. 173 and Fig. 5-20.)

Describe briefly the two principal models for the cooperative binding of ligands to proteins with multiple binding sites

Ans: In the concerted model, binding of a ligand to one site on one subunit results in an allosteric effect that converts all of the remaining subunits to the high-affinity conformation. As a result, all of the subunits are either in the low- or high-affinity conformation. In the sequential model, each subunit is changed individually to the high affinity conformation. As a result, there are many possible combinations of low- and high-affinity subunits.

Describe the concept of "induced fit" in ligand-protein binding.

Ans: Induced fit refers to the structural adaptations that occur when a ligand binds to a protein. This often involves a conformational change in the protein that alters the binding site to make it more complementary to the ligand.

Why is carbon monoxide (CO) toxic to aerobic organisms?

Ans: It binds to heme with a higher affinity than oxygen, and thus prevents oxygen from binding to hemoglobin.

For the binding of a ligand to a protein, what is the relationship between the Ka (association constant), the Kd (dissociation constant), and the affinity of the protein for the ligand?

Ans: Ka = 1/Kd. The larger the Ka (and hence the smaller the Kd), the higher the affinity of the protein for the ligand.

What is the chemical basis for the specificity of binding of an immunoglobin antibody to a particular antigen?

Ans: Specific binding results from complementarity between the chemical properties (such as size, charge, and hydrophobicity) of the antigen and the antigen-binding site of the antibody.

Explain briefly why the relative affinity of heme for oxygen and carbon monoxide is changed by the presence of the myoglobin protein.

Ans: The geometry of binding O2 and CO to heme is slightly different. In myoglobin there is a histidine residue that does not interact with the heme iron, but can interact with a ligand that is bound to the heme. It does not affect O2 binding but because of steric hindrance, it may prevent CO binding. As a result the relative affinity of protein-bound heme for CO and O2 is only 200, compared to 20,000 for free heme.

Explain why the structure of myoglobin makes it function well as an oxygen-storage protein whereas the structure of hemoglobin makes it function well as an oxygen-transport protein.

Ans: The hyperbolic binding of oxygen to the single binding site of myoglobin results in a high affinity even at the relatively low partial pressures of O2 that occur in tissues. In contrast, the cooperative (sigmoidal) binding of O2 to the multiple binding sites of hemoglobin results in high affinity at high partial pressures such as occur in the lungs, but lower affinity in the tissues. This permits hemoglobin to bind O2 in the lungs and release it in the tissues.

What properties of antibodies make them useful biochemical reagents? Describe one biochemical application of antibodies (with more than just the name of the technique).

Ans: The important properties are the high specificity of protein recognition, and the high affinity of the antibody-antigen association. These make possible immunoaffinity chromatography, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and immunoblotting, all of which are described on pp. 181-182.

Describe how immunoaffinity chromatography is performed.

Ans: The specific antibody is covalently attached to an inert supporting material, which is then packed into a chromatography column. The protein solution is passed through the column slowly; most proteins pass directly through, but those for which the antibody has specific affinity are adsorbed. They can subsequently be eluted by a buffer of low pH, a salt solution, or some other agent that breaks the antibody-antigen association.

An allosteric interaction between a ligand and a protein is one in which: A) binding of a molecule to a binding site affects binding of additional molecules to the same site. B) binding of a molecule to a binding site affects binding properties of another site on the protein. C) binding of the ligand to the protein is covalent. D) multiple molecules of the same ligand can bind to the same binding site. E) two different ligands can bind to the same binding site.

B

An individual molecular structure within an antigen to which an individual antibody binds is as a(n): A) antigen. B) epitope. C) Fab region. D) Fc region E) MHC site.

B

During muscle contraction, hydrolysis of ATP results in a change in the: A) conformation of actin. B) conformation of myosin. C) structure of the myofibrils. D) structure of the sarcoplasmic reticulum. E) structure of the Z disk.

B

The predominant structural feature in myosin molecules is: A) a b structure. B) an a helix. C) the Fab domain. D) the light chain. E) the meromyosin domain.

B

When oxygen binds to a heme-containing protein, the two open coordination bonds of Fe2+ are occupied by: A) one O atom and one amino acid atom. B) one O2 molecule and one amino acid atom. C) one O2 molecule and one heme atom. D) two O atoms. E) two O2 molecules.

B

Which of the following parts of the IgG molecule are not involved in binding to an antigen? A) Fab B) Fc C) Heavy chain D) Light chain E) Variable domain

B

A monoclonal antibody differs from a polyclonal antibody in that monoclonal antibodies: A) are labeled with chemicals that can be visualized. B) are produced by cells from the same organism that produced the antigen. C) are synthesized by a population of identical, or "cloned," cells. D) are synthesized only in living organisms. E) have only a single polypeptide chain that can recognize an antigen.

C

The fundamental cause of sickle-cell disease is a change in the structure of: A) blood. B) capillaries. C) hemoglobin. D) red cells. E) the heart.

C

A prosthetic group of a protein is a non-protein structure that is: A) a ligand of the protein. B) a part of the secondary structure of the protein. C) a substrate of the protein. D) permanently associated with the protein. E) transiently bound to the protein.

D

The amino acid substitution of Val for Glu in Hemoglobin S results in aggregation of the protein because of ___________ interactions between molecules. A) covalent B) disulfide C) hydrogen bonding D) hydrophobic E) ionic

D

The interactions of ligands with proteins A) are relatively nonspecific. B) are relatively rare in biological systems. C) are usually irreversible. D) are usually transient. E) usually result in the inactivation of the proteins.

D

Strutureal hange in hemoglobin from T to R

During tense state the porphyrin is concave and empty when O2 binds the ring is pulled centrically and becomes planar (flat)

Myoglobin and the subunits of hemoglobin have: A) no obvious structural relationship. B) very different primary and tertiary structures. C) very similar primary and tertiary structures. D) very similar primary structures, but different tertiary structures. E) very similar tertiary structures, but different primary structures.

E

Which of the following statements about protein-ligand binding is correct? A) The Ka is equal to the concentration of ligand when all of the binding sites are occupied. B) The Ka is independent of such conditions as salt concentration and pH. C) The larger the Ka (association constant), the weaker the affinity. D) The larger the Ka, the faster is the binding. E) The larger the Ka, the smaller the Kd (dissociation constant).

E

heme is bound in a pocket made largely from

E and F helices

______ in free heme could be oxidized to _____

Fe2+ Fe3+

Sickle-Cell mutation in hemoglobin is due to

Glu to Val substitution in B chain of HB

Two states that hemoglobin can exist in

Tense Relaxed

What is the structural basis for the complex binding curve of oxygen to hemoglobin?

Hemoglobin exists in more than one oxygen binding conformation. With a significant difference in the oxygen binding affinity

the affinity of hemoglobin to oxygen depends on (3)

O2 BPG pH

difference between O2 and CO binding to heme

O2 binds to heme with O2 axis at angle CO will bind to heme in a perpendicular arrangement

2, 3 BPG allows for ___________________ and ________________

O2 release in tissues adaption to changes in altitude

heme is a ________ compound

Organometallic

heme structure

Protophyrin ring and iron in ferrous state Fe2+

O2 binding trigger _________ conformational change

T -> R

conformational change from the T state to the R state involves

breaking ion pairs between the a1 and B2 interface

binding of CO to myoglobin is weakened by the fact that

the perpendicular arrangement is sterically hindered by distal His

Sigmoidal curve is therefore referred to as the

affinity transition curve

the concerted model is also called the all or none model because

all subunits are postulated to be the same conformation

hemoglobin is also a ______ protein

allosteric

Cooperativity is a special case of

allosteric regulation

ligand interactions are critical to life because they allow

an organism to respond rapidly and reversibly to changing metabolic and environmental circumstances

Why does Co bind over 20,000 times better?

because the carbon in Co has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+

sensitivity to O2 concentration allows hemoglobin to

bind O2 at higher pO2 (lungs) release oxygen at lower pO2 (tissue)

in an allosteric protein the __________ site is affected by a different part of the protein

binding

Allosteric protein

binding of a ligand to one site affects the binding properties of a different site on the same protein -can be positive or negative

in the sequential model each individual subunit....

can be in either form

Two models of cooperativity

concerted sequential

binding of O2 to one heme triggers _______ in remaining heme groups

conformational change

sigmoidal shape of hemoglobin curve represents

cooperative/allosteric nature of hemoglobin

Tense is ______ state Relaxed is _______ state

deoxy oxy

Some CO2 is simply exported by the tissues, the remaining CO2 is exported as _______________ formed by ________________ also producing a ________________

dissolved bicarbonate carbonic anhydrase proton

what prevents Fe2+ from turning into Fe3+

electron donating character of the coordinated nitrogen atoms

present in mM concentrations in _______

erythrocytes

Fe3+

ferric ion

Fe2+

ferrous ion

negative cooperativty

first binding even reduces affinity at remaining sites

positive cooperativity

first binding event increases affinity at remaining sites

R state is more

flexible

porphyrins are

groups of 4 pyrrole rings linked by methene bridges

circulating oxygen-binding protein

hemoglobin

Co blocks the function of (3)

hemoglobin myoglobin mitochondrial cytochromes that are involved in oxidative phosphorylation

aggregation that causes sickles in the red blood cells is due to

hydrophobic patch on Deoxy hemoglobin surface

when bpg is completely absent hemoglobin curve appears

hyperbolic

Fe is is bound in ________ and _______ to it

in plane perpendicular

the body will _____________ BPG production at high altitudes

increase

pH difference between lungs and metabolic tissues will

increase efficiency of O2 transport

CO is toxic to humans because

it binds to Fe in hemoglobin/myoglobin and prevents the binding of O2

if binding sites were not able to interact, what would be shape of curve?

linear

in both models the circle represents

low affinity

Tense state has ___________ for O2 Relaxed state has __________ for O2

lower affinity higher affinity

H+ ions will

lower the pH of the blood near the tissue (relative to the lungs)

prosthetic group

metal ion or organic compound that is covalently bound to a protein and is essential to its activity

Hemoglobin

multi-peptide protein that carries oxygen and CO2

in order to exhibit cooperativity binding sites....

must be able to interact with one another

main oxygen storage protein

myoglobin

2,3 BPG is _______regulator of Hb function

negatice

can Fe3+ bind to oxygen?

no

what occupies the 2 perpendicular bonds?

one end will bind the O2 the other will attach the heme group to an AA residue (His)

the binding pocket for BPG disappears upon

oxygenation

Hb affinity for oxygen depends on

pH

Fe-protoporphyrin IX is an example of a group of structures known as __________

porphyrins

cooperativity is _____ regulation

positive

2,3 BPG binds to

positively charged central cavity of Hb (B subunits)

Heme is a _____ group

prosthetic

each subunit of hemoglobin has ________ resulting in ______ O2 binding sites

prosthetic heme group 4

_________ decreases affinity for CO

protein pocket

______ lack affinity for O2

protein side chains

Iron in Ferrous state binds O2 ......

reversibly

increasing BPG or decreasing pH would shift Hemoglobin curve to the...

right

because of cooperative binding hemoglobin is more.....

sensitive to small differences in O2 between the tissues and lungs

thought to be one of the main adjustments that trigger T to R

shift in Helix F when O2 binds

2, 3 BPG

side product of glycolysis

shape of hemoglobin curve

sigmoidal

positive cooperativity results in

sigmoidal urve

tertiary structure of myoglobin

single polypeptide chain formed of helices and connected by turns -one oxygen binding site

Myoglobin

single polypeptide that carries oxygen and CO2

2,3 BPG is a __________, ________ charged molecule

small negatively

high affinity is represented by ____ in concerted and sequential models

square

2,3 BPG effect on Hb

stabilize T state

H+ will bind to Hb and ....

stabilize the T-state

when binding to heme in myoglobin why does CO adopt slight angle

steric hinderence from distal His

aggregation results in _________ that align into ________

strands insoluble fibers

Hemoglobin is a _______ protein

tetrameric

the 3d structure of each of the 4 hemoglobin subunits is similar to

the 3-d structure of myoglobin

Stabilization of T state die t proton binding is called

the Bohr effect

the additional protons produced by CO2 + H2O contribute to

the Bohr effect

The hemoglobin sigmoidal curve is a combination of

the high affinity state curve and low affinity state curve

negative charge of 2, 3 BPG is due to

the two phosphate groups

some ________ bind to o2 well but would generate ______ if free in solution

transition metals free radicals

stabilizing T-state results in

triggering O2 release