BSC 450: Chapter 5

Protein Function

5.1, Reversible Binding of a Protein to a Ligand: Oxygen-Binding

Proteins

Oxygen Can Bind to a Heme Prosthetic Groups

 Oxygen is important for mammals
 Problems about binding O2 in aqueous solution
o Certain metals can bind oxygen and can be used to help
o Fenton reaction – iron
 Others use copper – blue blood
 In order to use iron to transport and store oxygen, you need to
reduce its reactivity  heme
 Oxygen is poorly soluble in aqueous solutions and diffusion through tissues
is ineffective over even small distances
 Evolution of larger organisms depended on the evolution of proteins that
could store and transport oxygen
o No amino acids side chains are suited for reversible binding of O2
o Transition metals, such as iron and copper, can bind O2
 Multicellular organisms exploit metals, mainly iron, for oxygen transport
o Free iron promotes formation of reactive O2 species that can damage
DNA and macromolecules
o Iron used in cells is bound in forms that sequester it or make it less
reactive
o Iron is often incorporated into a protein-bound prosthetic group called
heme
 Heme consists of a complex ring structure called protoporphyrin and a
bound iron atom in its ferrous state (Fe2+) – binds oxygen reversibly
o The iron atom has six coordination bonds, four to nitrogen atoms in
the flat porphyrin ring, and two perpendicular to the porphyrin
o Coordinated nitrogen atoms help prevent conversion of heme iron to
the ferric (Fe3+) state which does not bind oxygen
o Heme is in many oxygen transporting proteins and other proteins such
as the cytochrome that participate in redox reactions
 Free heme molecules (not bound to protein) leave Fe2+ with two open
coordination bonds
 o Reaction of one O2 with two free heme molecules can result in
irreversible conversion of Fe2+ to Fe3+
 Fe2+ binds oxygen, Fe3+ does not
o Prevented in heme-containing proteins by sequestering each heme
deep within the protein structure – access to two open coordination
bonds is restricted
 In gobins, one coordination bond is occupied by a side chain nitrogen of a
His residue (the proximal His), the other a binding site for molecular O2
o When oxygen binds, the heme properties change (purple to red blood)
 Some smaller molecules (CO2, NO) coordinate to heme iron with greater
affinity than does O2
o When a molecule of CO is bound to heme, O2 is excluded, which is
why CO is highly toxic to aerobic organisms
 By surrounding and sequestering heme, oxygen-binding proteins regulate the
access of small molecules to the heme iron

Globins Are a Family of Oxygen-Binding Proteins

 Globins: widespread family of proteins, all with similar primary and tertiary
structures – in eukaryotes and some bacteria
o Most function in oxygen transport or storage, or sensing O2, NO, CO2
o 33 kinds of globins in C elegans – the simple nematode worm
o 4 kinds in humans and other mammals
 Myoglobins: monomeric, facilitates oxygen diffusion in muscle tissue
o Abundant in muscles of diving marine mammal – also has oxygen
storing function for prolonged excursions under the sea
o Has a heme binding pocket
 Hemoglobin: tetrameric, responsible for oxygen transport in the bloodstream
 Neuroglobin: monomeric, prevents brain from hypoxia or ischemia
 Cytoglobin: monomeric, found in walls of blood vessels, regulates NO
levels

Myoglobin Has a Single Binding Site for Oxygen

 Single polypeptide of 153 amino acid residues with one heme molecules
 8 a-helical segments connected by bends (contain 75% of aa’s in the protein)
 Helical segments are named A through H
 Individual amino acid residues are designated wither by position in sequence
or location on particular a-helical segment
 o Ex: His residue coordinated to heme in myoglobin is called His93 or
His F8
 Bends are designated AB, CD, EF, FG, reflecting the a-helical segments
they connect

Protein-Ligand Interactions Can Be Described Quantitatively

 Simple equilibrium expressions can typically describe reversible binding of
protein to a ligand
 Ka, association constant: describes the equilibrium between the complex and
the unbound components of the complex
o Measure of the affinity of the ligand for the protein
o Higher Ka, greater affinity of ligand for the protein
 Ka is also equivalent to the ratio of the rates of the forward and reverse
reactions that form the PL complex
o Rate constants are proportionality constants: association rates are
described by ka and kd (lowercase k)
o First order reaction: involves one molecule, PL  P + L
 kd unit is s-1
o Second order reaction: involves two molecules, P + L  PL
 ka unit is M-1s-1
 The ratio of bound to free protein is directly proportional to the
concentration of the free ligand
o When ligand concentration is greater than ligand-binding site
concentration, binding of ligand does not appreciably change the free
ligand concentration
 Y: the fraction of ligand-binding sites on the protein that are occupied by
ligand (Y = binding sites occupied/total binding sites = [PL]/[P]+[L]
o Y is a hyperbolic function of [L]
o Fraction of ligand binding sites occupied approaches saturation
asymptomatically as [L] increases
o The [L] at which half of the available ligand-binding sites are
occupied corresponds to 1/Ka
 Kd, the dissociation constant: the reciprocal of Ka, has units of molar
concentration (M), is the equilibrium constant for the release of ligand
o [L] = Kd, half of ligand binding sites are occupied
o [L] falls below Kd, less protein has ligand bound to it
o [L] is 9x greater than Kd, 90% of sites will be occupied
o Lower Kd  higher affinity of ligand for a protein
 o Kd is equivalent to the molar concentration of ligand at which half of
the available ligand binding sites are occupied (half saturation)
o The more tightly a protein binds to a ligand, the lower concentration
needed for half the sites to be occupied, the lower the Kd
 Binding of oxygen to myoglobin follows these patterns, but because O2 is a
gas, binding is expressed in terms of partial pressure, so the equation must
be adjusted to Y = [O2]/[O2]+Kd
o Kd equals the [O2] where half of the available ligand binding sites are
occupied, so Y=[O2]/[O2]+[O2]0.5
o When oxygen is used as a ligand, the partial pressure is varied, it’s
easier to measure than the concentration of oxygen gas in solution
 The concentration of a volatile substance in solution is always
proportional to the local partial pressure of the gas
 Y=pO2/pO2+p50
o Oxygen binds tightly to myoglobin with a P50 of only 2 torr

Protein Structure Affects How Ligands Bind

 Binding of a ligand to a protein is greatly affected by protein structure and is
often accompanied by conformational changes
o Ex: the relative affinity of CO2 and O2 for heme changes when it is
bound to a globin because of the globin structure
 When heme is bound to myoglobin, the presence of distal His increases its
affinity for O2
o The Fe-O2 complex is more polar than the Fe-CO complex
o Partial oxidation of the iron atom distributes a partial negative charge
across oxygen atoms in the bound O2
o Imidazole side chain hydrogen bonds with O2, stabilizing the polar
complex electrostatically
o Affinity for O2 is increased by a factor of 500 – no effect for Fe-CO
binding in myoglobin
o The 20,000 fold binding affinity of free heme for CO compared with
O2 declines to 40 fold for heme embedded in myoglobin
 This electrostatic effect on O2 binding is even more dramatic in some
invertebrate hemoglobin
o Two groups in the binding pockets can form strong hydrogen bonds
with O2 – causing heme to bind O2 with greater affinity than CO
o Selective enhancement of O2 affinity helps prevent CO poisoning
generated by heme catabolism
  Binding of O2 to heme in myoglobin also depends on molecular motions -
“breathing” in the protein structure
o Heme is buried, so rapid molecular flexing of amino acid side chains
producing cavities for O2 to move in and out
o Distal His acts as a gate to control access to one major pocket near the
heme iron
 In some other globins, the distal His is directly coordinated with
the heme iron at the location where ligands must bind
 In these globins, the O2 or ligand must replace the distal His to
bind – with a hydrogen bond forming between the distal his and
O2 after binding occurs

Hemoglobin Transports Oxygen in the Blood

 Hemoglobin in erythrocytes (red blood cells) carries oxygen in the blood
o Small, biconcave discs formed from precursor stem cells called
hemocytoblasts
o The stem cells produce daughter cells that form large amounts of
hemoglobin and lose their organelles
o Erythrocytes are incomplete, vestigial cells, unable to reproduce, and
can only survive 120 days in humans
o They function to carry hemoglobin which is dissolved in cytosol at
high concentrations
 In arterial blood passing from lungs through the heart to the peripheral
tissues, hemoglobin is 96% saturated with oxygen
 In venous blood returning to the heart it is only 64% saturated
 Each 100mL blood passing through tissue releases 1/3 O2 it carries
 Myoglobin is relatively insensitive to small changes in concentration of
dissolved oxygen and so it functions well as an oxygen-storage protein
o Hyperbolic binding curve for oxygen
 Hemoglobin has multiple subunits and O2 binding sites, and is better suited
for oxygen transport
o Interactions among hemoglobin subunits cause conformational
changes that alter the affinity of the protein for oxygen
 The modulation of oxygen binding allows the O2 transport protein to
respond to changes in oxygen demand by tissues V
 Oxygenated blood is lighter, non-oxygenated is darker

Hemoglobin Sub-Units Are Structurally Similar to Myoglobin

 Hemoglobin is roughly spherical
  Tetrameric with four heme prosthetic groups, one associated with each
polypeptide chain
o Tetramer of two subunits (a2B2)
o Each subunit is similar to myoglobin (contains a heme)
 Myoglobin and hemoglobin are similar
o The 3D structures are similar – enough overlap in amino acid residues
to product similar 3D residues
 Two types of globin: a chains and B chains
o Different sequences, but similar structures to each other and to
myoglobin
 A subunit lacks the short D helix that myoglobin has
 The heme binding pocket is made up largely of the E and F helices in each
of the subunits
 The quaternary structure of hemoglobin features strong interactions between
unlike subunits
o a1B1 and a2B2 involve more than 30 residues, with strong interaction
o However, the treatment of hemoglobin with urea tends to disassemble
the tetramer into aB dimers
o Hydrophobic effect plays the major role in stabilizing these interfaces
o Also, many hydrogen bonds and a few ion pairs/salt bridges

Hemoglobin undergoes a Structural Change on Binding Oxygen

 Two major confirmations of hemoglobin: R and T state
 Oxygen can bind to either state, but has a higher affinity for the R state
o Binding stabilizes the R state
 Without oxygen present, the T state is more stable – predominant
conformation of deoxyhemoglobin
o T state is stabilized by a greater number of iron pairs, many at the
a1B2 interface
o Binding of O2 to hemoglobin in the T state triggers a conformational
change to the R state
 When proteins undergo this transition, the individual subunit structures only
change a little, but the aB subunits slide past each other, rotating and
narrowing the pocket between the B subunits
o Some of the ion pairs that stabilize the T state are broken and new
ones form
 Max Perutz proposed that the T  R transition is triggered by the changes in
the positions of key amino acid side chains surrounding them heme
 o In T state, porphyrin is slightly puckered, causing the heme to slightly
protrude on the proximal His side
o Binding of O2 causes the heme to assume a more planar confirmation,
shifting the position of the proximal His and the attached F helix
o These changes lead to adjustments in the iron pairs at the a1B2
interface
o Proximal his residue allows it to only interact with one oxygen

Hemoglobin Binds Oxygen Cooperatively

 Hemoglobin must bind oxygen in the lungs where pO2 = 13.3
 Hemoglobin must release oxygen in the tissues where pO2 =. 4
 Myoglobin, or another oxygen binding protein with a hyperbolic curve
would be ill-suited for this function
o If the protein that bound O2 with high affinity would bind it
efficiently in the lungs but would not release much of it in the tissues
o If the protein bound oxygen with low affinity to release it in the
tissues, it would not pick up much oxygen in the lungs
 Hemoglobin solves this problem by undergoing a transition from a low
affinity (T) state to a high affinity (R) state as more O2 is bound
o Hybrid, S-shaped, sigmoid, binding curve for oxygen
 A single subunit protein with a single ligand binding site cannot produce a
sigmoid curve – even if there is a conformational change – because each
ligand binds independently and does not affect binding of other molecules
 O2 binding to individual subunits of hemoglobin can alter affinity for O2 in
adjacent subunits
o The first molecule of O2 binds weakly with deoxyhemoglobin in the
T state
o Its binding leads to conformational changes communicated to other
subunits that make it easier for additional O2 to bind
o The T  R conformation occurs more readily in the second subunit
once O2 is bound to the first subunit
o The last (fourth) O2 molecule binds to a heme in a subunit that is
already in the R state, and binds with much higher affinity than the
first
 Allosteric protein: one in which the binding of a ligand to one site affects the
binding properties of another site on the same protein (can be positive or
negative)
 o Ligand/modulators induce other shapes/conformations that are
converted to more active or less active forms of the protein
o Modulators can be inhibitors or activators
o Homotropic: when normal ligand and modular are identical
 Normal ligand of the protein is the allosteric regulator
o Heterotropic: when modulator is a molecule other than the normal
ligand
 Different ligand affects binding of the normal ligand
o Some proteins have two or more modulators – can have both
homotropic and heterotropic interactions
o Cooperativity in Hb = positive homotropic regulation
 Cooperative binding of a ligand to a multimeric protein (ex: binding of O2
to hemoglobin) is a form of allosteric binding
o O2 is a ligand and an activating homotropic modulator
o Only one o2 binding site on each subunit, so the allosteric affects
giving rise to cooperativity are mediated by conformational changes
transmitted from one subunit to another by subunit-subunit
interactions
o Sigmoid binding curve is diagnostic of cooperative binding
 More sensitive response to ligand concentration
 Important to the function of many multi-subunit proteins
o Depend on variations in structural stability of different parts of a
protein
 Binding site of allosteric protein typically consists of stable
segments in proximity to unstable segments capable of frequent
changes in conformation or intrinsic disorder
 When ligand binds, moving parts of protein’s binding site may
be stabilized in a particular conformation, affecting the
conformation of adjacent polypeptide subunits
 If the entire binding site were highly stable, then few structural
changes could occur in this sit or be prorogated to other parts of
the protein when a ligand bound
 As with myoglobin, ligands other than oxygen can bind to hemoglobin
o Ex: carbon monoxide – binds to hemoglobin about 250 times better
than does oxygen
 Critical hydrogen bond between O2 and distal His is not as
strong in human hemoglobin as in most mammalian myoglobin,
so binding of O2 relative to CO is not augmented quite as much
  CO has similar size and shape to O2 – it can fit to the same
binding site
 Small dipole moment in CO, C is more negative, O is more
positive
 CO binds free heme over 20,000 times better than O2
 The C in CO has a filled lone electron pair that can be
donated to vacant d-orbitals on the Fe2+
 C-O-Fe2+ in a linear geometry for free heme
o Human exposure to CO can have tragic consequences
 It blocks the function of myoglobin, hemoglobin, and
mitochondrial cytochromes that are involved in oxidative
phosphorylation
o In globins, CO binds to heme only 250 times better than O2
o Cyanide poisoning

Cooperative Ligand Binding Can Be Described Quantitatively

 Reversible, transient process of chemical equilibrium
o A + B  AB
o Ligand: a molecule bound reversibly by a protein
 Typically a small molecule, H20 not included until special
o Binding site: a region in the protein where the ligand binds,
complementary to the ligand size, shape, charge, hydrophobic or
hydrophilic character
o The interaction is highly specific
o Binds via noncovalent interactions – allows to be transient
o Proteins can have conformational changes
o Induced fit: the structural adaptation occurs between protein and
ligand
 Protein conformational changes may occur upon ligand binding
 This adaptation is called the induced fit
 Induced fit allows for tighter binding of the ligand
 Induced fit allows for high affinity for different ligands
 Binding: Quantitative description
o kinetics
 Consider a process in which a ligand (L) binds reversibly to a
site in the protein (P)
 The kinetics is described by:
 Association velocity and dissociation velocity
 o Association reaction is a second order reaction
 va=ka[P][L]
 ka, association rate constant
o Dissociation reaction is a first order reaction
 vd=kd[PL]
 kd, dissociation rate constant
o When the process reaches equilibrium
 The association and dissociation rates are
equal
 v=ka[P][L]=kd[PL]
o Thermodynamics
 At equilibrium
 The binding (association) equilibrium is described by Ka
(association constant)
o Ka = [PL]/[P][L] = ka/kd
 The dissociation equilibrium is described by Kd
(dissociation constant)
o Kd = [P][L]/[PL] = kd/ka = 1/Ka
o Graphical analysis
 Ligand concentration is known: [L] = [L]total
 Kd can be determine by graphically or via least-squares
regression
 Cooperative binding of oxygen by hemoglobin was first analyzed by
Archibald Hill in 1910
o Cooperative proteins have multiple binding sites
 So Ka becomes Ka = [PLn]/[P][L]n
 Theta becomes tehta = [L]n/[L]n+Kd
o The Hill equation: log (Y / 1-Y) = n log [L] – log Kd
o Hill plot: plot of log[Y/(1-Y)] vs log [L]
o So the hill plot should have a slope of n ? no…
 Experimentally determined slope represents the degree of
interaction between binding sites – not number
 nH denotes the slot of a hill plot – the Hill coefficient, which is a
measure of the degree of cooperativity
 If nH = 1, ligand binding is not cooperative, subunits do
not communicate
 If nH > 1, positive cooperativity in ligand binding –
binding of one molecule of ligand facilitates the binding
of others (ex: hemoglobin and oxygen)
  If nH = n, it has reached its theoretical upper limit –
binding is completely cooperative, all binding sites on
protein would bind ligand simultaneously and no protein
molecules partially saturated with ligand would be
present under any conditions
o Never reached in practice, nH always less than n
 If nH < 1, negative cooperativity, binding of one molecule
of ligand impedes the binding of others, rare
o To adapt hill equation to the binding of oxygen, substitute pO2 for [L]
and p50n for Kd
o Hill plot of cooperativity

Two Models Suggest Mechanisms for Cooperative Binding

 Much to be learned about how the T  R transition occurs (2 models)
o T = tense state
 More interactions between subunits, more stable
 Lower affinity for O2
o R = relaxed state
 Fewer interactions, more flexible
 Higher affinity for O2
o O2 binding triggers conformational change from T to R state, Hb
binding affinity to O2 increases
 Conformational change is triggered by oxygen binding
 MWS Model/Concerted Model
o Assumes:
 That the subunits of a cooperatively binding protein are
functionally identical
 That each subunit can exist in at least two conformations
 All subunits undergo the transition from one conformation to
the other simultaneously
o No protein has individual subunits in other conformations
o The two conformations are in equilibrium
o The ligand can bind to either conformation, but binds more tightly to
the R state
o Successive binding of ligand molecules to the low-affinity
conformation makes a transition to the high-affinity conformation
more likely
 Sequential Model
 o Ligand binding can induce a change of conformation in an individual
subunit
o A conformation change in one subunit makes a similar change in an
adjacent subunit, as well as making the binding of a second ligand
molecule more likely
o Two more potential intermediate states in this model than the
concerted model
 The two models are not mutually exclusive, the concerted model may be
viewed as the “all-or-none” limiting case of the sequential model
 Cooperativity
o Must be a protein with multiple binding sites
o Binding sites must be able to interact with each other (not directly)
o Positive cooperativity
 First binding event increases binding affinity of remaining sites
 Recognized by sigmoidal binding curves
o Negative cooperativity
 First binding event reduces binding affinity of remaining sites
o O2 binding the first binding site triggers T  R conformational
change

Hemoglobin Also Transports H+ and CO2

 In addition to carrying all the oxygen required by cells from the lungs to the
tissues, hemoglobin carries two end products o cellular respiration, H+ and
CO2 from the tissues to the lungs and kidneys where they are excreted
 The CO2 produced by oxidation of organic fuels in mitochondria is hydrated
to form bicarbonate. HCO3-
o This reaction is catalyzed by carbonic anhydrase – abundant in
erythrocytes
o CO2 is not very soluble and bubbles would form in tissues and blood
if it were not converted to bicarbonate
o This hydration reaction results in an increase in the H+ concentration
and a decrease in pH in the tissues
o Binding of oxygen by hemoglobin is profoundly influences by pH and
CO2 concentration
 Interconversion of CO2 is of great importance to the regulation
of oxygen binding and release in the blood
 Hemoglobin transports 40% of the total H+ and 15-20% of the total CO2
formed in the tissues to the lungs and kidneys
 o Binding of H+ and CO2 is inversely related to the binding of oxygen
 At low pH and high CO2 concentration of peripheral tissues,
affinity of hemoglobin for oxygen decreases as H+ and CO2 are
bound and O2 is released to the tissues
 In the capillaries of the lung, as CO2 is excreted and blood pH
consequently rises, the affinity of hemoglobin for oxygen
increases and the protein binds more O2 for transport to the
peripheral tissues
o Bohr effect: the effect of pH and CO2 concentration on the binding
and release of oxygen by hemoglobin
 HHb+ + O2  HbO2 + H+
 The O2 saturation curve is influenced by the H+ concentration
 Both O2 and H+ are bound by hemoglobin, but with inverse
affinity
 When oxygen concentration is high, as in the lungs,
hemoglobin binds O2 and releases protons
 When oxygen concentration is low, as in the peripheral
tissues, H+ is bound and O2 is released
 Actively metabolizing tissues generate H+ and CO2
 CO2 +H20  H+ + HCO3- by carbonic anhydrase
 Increases [H+] of the blood near the tissues relative to the
lungs
 Bohr effect: the stimulation of oxygen release by CO2
and H+
o Metabolic Acidosis - normal blood pH is around 7.4, acidosis occurs
when pH 7.35 or lower - rapid or shallow breathing, confusion,
fatigue, headache, sleepiness, lack of appetite, increased heart rate,
breath smells fruity (which is a sign of diabetic acidosis)
 Oxygen and H+ are not bound at the same sites in hemoglobin
o Oxygen binds to the iron atoms of hemes
o H+ binds to any of the amino acid residues in the protein
o His146 (His HC3)makes a major contribution to the Bohr effect
 When protonated, this residue forms on of the iron pairs to
Asp94 that helps to stabilize deoxyhemoglobin in the T state
 Stabilizes the protonated from of His HC3, giving this residue a
high pKa in the T state
 H+ binds to Hb and stabilizes the T state
 Added proton strengthens interactions
  The pKa falls to its normal 6.0 in the R state because the iron
pair cannot form
 This residue is largely unprotonated in oxyhemoglobin at
pH 7.6 (lungs)
 As concentration of H+ rises, protonation of His HC3 promotes
release of oxygen by favoring a transition to the T state
 Protonation of the a-terminal residues of the a-subunits, certain
other His residues, and perhaps other groups have a similar
effect
o The four polypeptide chains of hemoglobin communicate not only
about O2 binding to their heme groups but also about H+ binding to
specific amino acid residues
o Hemoglobin also binds CO2 in a manner inversely related to the
binding of oxygen
 It bonds as a carbamate group to the a-amino acids at the a-
terminal end of each globin chain, forming
carbaminohemoglobin
 This reaction produces H+, contributing to the Bohr effect
 Bound carbamates also form additional salt bridges that help to
stabilized the T state and promote the release of oxygen
 When the concentration of carbon dioxide is high, in peripheral tissues,
some CO2 binds to hemoglobin and the affinity for O2 decreases, causing its
release
 When hemoglobin reaches the lungs, the high oxygen concentration
promotes binding of O2 and release of CO@

Oxygen Binding to Hemoglobin Is Regulated by 2,3-Biphosphoglycerate

 The interaction of 2,3-biphosphoglycerate (BPG) with hemoglobin
molecules further refines the function of hemoglobin, and is an example of
heterotopic allosteric modulation
 2,3-BPG
o Small negatively charged molecule
o Binds to the positively charged central cavity of Hb
o Only one 2,3-BPG per Hb
 Stabilizes the T state
 Promotes O2 release
 Negative heterotrophic regulator of Hb function
o Present at mM concentrations in erythrocytes
 BPG is present in high concentrations in erythrocytes
 o Hemoglobin contains substantial amounts of BPG when isolated,
which are difficult to remove completely
o O2 binding curves discussed up to this point have been obtained in the
presence of BPG
 BPG is known to greatly reduce the affinity of hemoglobin for oxygen –
inverse relationship between the binding of O2 and the binding of BPG
 BPG binds at a site distant from the oxygen-binding sire and regulates the
O2 binding affinity of hemoglobin in relation to the pO2 in the lungs
o BPG is important in the adaptation to the lower pO2 at high altitudes
 At sea level, the binding of O2 to hemoglobin is regulated such that the
amount of O2 delivered to the tissues is nearly 40% of the maximum that
could be carried by the blood
 At a higher altitude, where the pO2 is considerably lower, the delivery of O2
to the tissues is now reduced
o After a few hours at the higher altitude, the BPG concentration in the
blood has begun to rise, decreasing hemoglobin affinity for oxygen
o This has a small effect on binding O2 in the lungs, but a great effect
on releasing O2 in the tissues
o As a result, the delivery of oxygen to the tissues is resorted to nearly
40% of the O2 that can be transported by the blood
o The situation is reversed when the person is returned to seal level
 The BPG concentration in erythrocytes also increases in people suffering
from hypoxia, lowered oxygenation of peripheral tissues due to inadequate
functioning of the lungs or circulatory system
 The site of BPG binding to hemoglobin the cavity between the B subunits in
the T state
o This cavity is lined with positively charged amino acid residues that
interact with the negatively charged groups of BPG
o Unlike O2, only one molecule of BPG is bound to each hemoglobin
tetramer
 BPG lower’s hemoglobin’s affinity for oxygen by stabilizing the T state
o The transition to the R state narrows the binding pocket for BPG,
precluding BPG binding
 In the absence of BPS, hemoglobin is converted to the R state more easily
 Regulation of oxygen binding to hemoglobin by BPS has an important role
in fetal development
 o A fetus must extract oxygen from its mother’s blood, so fetal
hemoglobin must have greater affinity than the maternal hemoglobin
for O2
o The fetus synthesized gamma subunits rather than beta subunits,
forming a2g2 hemoglobin
o This tetramer has a much lower affinity for BPG than normal adult
hemoglobin, and a higher affinity for O2

Sickle Cells Anemia Is a Molecular Disease of Hemoglobin

 Sickle cell anemia demonstrates the importance of amino acid sequence in
determining the secondary, tertiary, and quaternary structures of globular
proteins, and their biological functions
 Sickle cell anemia is due to a mutation in hemoglobin
o Glu 6 -> Val in the B chain of Hb
o Formation of hydrophobic surface
o The new Val chain can bind to a different Hb molecule to form a
strand
o This sickles the red blood cells
o Untreated homozygous individuals generally die in childhood
 Heterozygous individuals exhibit a resistance in malaria
 Over 500 genetic variants of hemoglobin are known to occur in the human
population
o All but a few are rare
o Most variants consist of a single amino acid difference
o Effects are often minor but can be extraordinary
 Each hemoglobin variation is the product of an altered gene
o Alleles: variant genes
 Homozygous: two copies of an allele
 Heterozygous: one copy of each of two different alleles
 Sickle cell anemia occurs when an individual inherits the allele for sickle
cell hemoglobin from both parents
o The erythrocytes of these individuals are fewer and abnormal
o In addition to a large number of immature cells, the blood contains
many long, thin, sickle-shaped erythrocytes
o When hemoglobin from sickle cells (hemoglobin S/HbS) is
deoxygenated, it becomes insoluble and forms polymers that
aggregate int tubular fibers
  Normal hemoglobin (hemoglobin A/HbA) remains soluble
when deoxygenated
o The insoluble fibers of the deoxygenated HbS cause the deformed,
sickle shape of the erythrocytes and the proportion of sickled cells
increases greatly as blood is deoxygenated
 The altered properties of Hbs results from a single amino acids substitution,
a Val instead of a Glu reside at position 6 in the two B chains
o The R groups of valine has no electric charge
o Glutamate has a negative charge at pH 7.4
o HbS has two fewer negative charges than HbA
o This creates a sticky hydrophobic contact point at position 6 of the B
chain, which is the outer surface of the molecules
 Causes deoxyHbS molecules to associate abnormally with each
other, forming the long, fibrous aggregates
 Sickle cell anemia is life threatening and painful
o Weakness, dizziness, shortness of breath, heart murmurs, and
increased pulse rate
o Hemoglobin content is only half the normal value, because sickled
cells are very fragile and rupture easily  resulting in anemia
o The capillaries become blocked by the long, abnormal cells, causing
severe pain and interfering with normal organ function (major factor
of early death for many who have the disease)
 Without treatment, people usually die in their childhood
 Frequency of the allele is high in Africa
o When heterozygous, the allele confers a small but significance
resistance to lethal forms of malaria
 Heterozygous individuals experience a milder condition –
sickle-cell trait – only about 1% of their erythrocytes become
sickled on deoxygenation
o May live normal lives if they avoid vigorous exercise and other
stresses on their circulatory system
o Natural selection has resulted in an allele population that balances the
deleterious effects of the homozygous condition against the resistance
to malaria afforded by the heterozygous condition

5.2, Complementary Interactions Between Proteins and Ligands:

The Immune System and Immunoglobins
Immune Response: an intricate and coordinated set of interactions among many
classes of proteins, molecules, and cell types

The Immune Response Includes a Specialized Array of Cells and Proteins

 Leukocytes (white blood cells) bring immunity
o Macrophages
o Lymphocytes
o Develop from undifferentiated stem cells in the bone marrow
o Can leave the bloodstream to patrol tissues
 Each cell produces proteins that can recognize and bind
molecules that might signal an infection
 The immune response consists of two complementary systems
o The humoral immune system
 Directed at bacterial infections and extracellular viruses
 Can also respond to individual foreign proteins
o Cellular immune system
 Destroys host cells infected by viruses
 Destroys some parasites and foreign tissues
 At the heart of the humoral immune response are proteins called
antibodies/immunoglobins (Ig)
o They bind bacteria, viruses, or large molecules identified as foreign
and target them for destruction
o Make up 20% of blood protein
o Produced by B lymphocytes (B cells) – complete their development in
the bone marrow
 At the heart of the cellular immune response are a class of T lymphocytes (T
cells) known as cytotoxic T cells
o T-cell receptors on the surface of T cells recognize infected
cells/parasites
o Receptors are proteins, usually on the outer surface of cells and extend
through the plasma membrane that recognize and bind extracellular
ligands, triggering changes within the cell
 Helper T cells function to produce soluble signaling proteins called
cytokines, including interleukins
o They interact with macrophages
o Participate indirectly in destruction of infected cells and pathogens
 Stimulating selective proliferation of Tc and B cells that can
bind a particular antigen – clonal selection
 o Clonal selection: increases the number of immune system cells that
can respond to a particular pathogen
o Th cells are the primary targets of the HIV infection
 Elimination of these cells incapacitates the entire immune
system
 Each recognition protein of the immune system, either a T cell receptor or an
antibody produced by a B cell binds a particular chemical structure
o Humans can produce 10^8 different antibodies with distinct binding
specificities
o So any chemical structure on the surface of a virus will likely be
bound by one or more antibodies
o Antibody diversity is derived from random reassembly of a set of
immunoglobin gene segments through genetic recombination
mechanisms
 Antigen: any molecule or pathogen capable of eliciting an immune response
is called an antigen
o May be a virus, bacterial cell wall, or an individual protein or
macromolecules
o Complex antigens may be bound by several different antibodies
o An individual antibody or t-cell receptor binds only a particular
molecular structure within the antigen, called its antigenic determinant
or epitope
 Epitope: the part of the antigen recognized by the antibody
 It would not be productive for the immune system to respond to small
molecules that are common intermediates and products of cellular
metabolism
o But when small molecules are covalently attached to large proteins in
the lab, they can be used to elicit an immune response
o these small molecules attached to bigger elements are called haptens
 MW below 5,000 – attached to bigger elements
 Immune system would then recognize hapten – and then when
your body develops memory, when exposed just to the small
molecule it will remember just the small molecule because you
already presented it in the form of a hapten
o the antibodies produced in response to protein-linked haptens will
bind to the same molecules in their free form

Antibodies Have Two Identical Antigen-Binding Sites

 Immunoglobulin G (IgG) is the major class of antibody molecule and one of
the most abundant proteins in the blood serum
o IgG has four polypeptide chains
 Two heavy chains: large ones
 Two light chains
 Linked by noncovalent disulfide bonds into a complex
 The heavy chains interact at one end, then branch to interact
separately with the light chains  Y shaped molecules
o At the “hinges: separating the base of an IgG molecule from its
branches, the immunoglobin can be cleaved with proteases
 Liberates the basal fragment (Fc because it crystallizes readily)
and the two branches (Fab – the antigen binding fragments)
 Each branch has a single antigen-binding site
 Structure of immunoglobulins was established by Edman and Porter
o Each chain is made up of identifiable domains
 Some constant in sequence and structure from one IgG to the
next; others variable
o Immunoglobulin fold: the characteristic structure of constant domains
 A well conserved structural motif in the all-B class of proteins
o Three constant domains in each heavy chain
o One constant domain in each light chain
o One variable chain each – most of the variability in amino acid
sequence is found here
 Variable domains associate to create the antigen-binding sire
 Allowing formation of an antigen-antibody complex
 IgG is one of five classes of immunoglobulins
o Each class has a characteristic type of heavy chain – denoted alpha,
delta, epsilon, gamma, and mu for IgA, IgD, IgE, IgG, and IgM
 two types of light chain occur in all classes
o IgD and IgE are similar in overall structure to IgG
o IgM occurs in either monomeric, membrane-bound form, or in
secreted form that is a cross-linked pentamer of this basic structure
 The first antibody to be made by B lymphocytes and the major
antibody in the early stages of a primary immune response
 Some B cells begin to produce IgD, but the particular
function of IgD is less clear
o IgA is found in secretions such as saliva, tears, and milk, and can be a
monomer, dimer, or trimer
 o IgG is the major antibody in secondary immune responses – which are
initiated by a class of B cells called memory B cells
 Part of organism’s ongoing immunity to antigens already
encountered and dealt with
 Most abundant immunoglobulin in the blood
 When IgG binds to an invading bacterium or virus, It activates
certain leukocytes such as macrophages to engulf and destroy
the invader
 It also activated some other parts of the immune response
 Receptors on the macrophage surface recognize and bind the Fc
region of IgG
 When these Fc receptors bind an antibody-pathogen complex,
the macrophage engulfs the complex by phagocytosis
 Phagocytosis of an antibody-bound virus by a
macrophage
o IgE plays an important role in the allergic response, interacting with
basophils in the blood and with histamine-secreting cells called mast
cells – widely distributed in tissues
 This immunoglobulin binds through its Fc region to special Fc
receptors on the basophils or mast cells
 In this form, IgE serves as a receptor for antigen
 If antigen is bound, the cells are induces to secrete histamine
and other active amines that cause dilation and increased
permeability of blood vessels
 These effects on the blood vessels are thought to facilitate the
movement of the immune system cells and proteins to the sites
of inflammation
 They also produce the symptoms normally associated with
allergies
 Pollen or other allergens are recognized as foreign,
triggering an immune response normally reserved for
pathogens

Antibodies Bind Tightly and Specifically to Antigen

 Binding specificity of antibody is determined by amino acid residues in
variable domains of heavy and light chains
o Many are variable but not equally so, some are hypervariable –
especially likely to vary (such as those lining the antigen-binding site)
  Specificity is conferred by chemical complementarity between antigen and
specific binding site – in terms of shape and location of charged, nonpolar,
and hydrogen bonding groups
o Ex: binding site with a negatively charged group may bind an antigen
with a positive charge in the complementary position
o In many cases, complementarity is achieved interactively as the
structures of antigen and binding site influence each other as they
come closer together
 Conformational changes in the antibody and/or antigen then allow the
complementary groups to interact fully – example of induced fit
o When antigen binds to antibody, binding cavity changes
 Without antigen  with antigen, cavity becomes larger
 Hydrophobic interactions and hydrogen bonds strength
interaction between antigen and antibody
 A typical antibody-antigen interaction is quite strong
o Characterized by Kd values as low as 10^-10
o The Kd reflects the energy derived from the hydrophobic effect and
the various ionic, hydrogen bonding, and van der Waals interactions
that stabilize the binding
 The binding energy to produce a Kd of 10^-10 is about 65
kJ/mol

The Antibody-Antigen Interaction Is the Basis for a Variety of Important

Analytical Procedures
 The extraordinary binding affinity and specificity of antibodies makes them
valuable analytical reagents
 Two types of antibody preparations are in use:
o Polyclonal antibodies
 Those produced by many different B lymphocytes responding
to one antigen
 Ex: protein injected into an animal
 Cells in the population of B lymphocytes produce antibodies
that bind specific, different epitopes within the antigen
 Epitope: part of antigen recognized by antibody
 Contains a mixture of antibodies that recognize different parts
of the protein
o Monoclonal Antibodies
 Synthesized by a population of identical B cells (a clone) grown
in cell culture
  Homogenous, all recognizing the same epitope
 Technique by Milstein and Kohler
 Large amounts of antibodies of nearly and desired
specificity can be obtained by fusing a short-lived
antibody-producing cell with an immortal myeloma cell
 A mixture of plasma cells from this spleen is fused in
vitro with myeloma cells – each of the resulting hybrid
cells called hybridoma cells – indefinitely produces the
identical antibody specified by the parent cell from the
spleen
 The hybridoma method of producing monoclonal
antibodies had a dramatic impact on biology and
medicine
o Monoclonal antibodies attached to solid supports
can be used as affinity columns to purify scarce
proteins, this method has been used to purify
interferon (an antiviral protein) 5000-fold from
crude mixture
o The detection in blood of isozymes that are
normally localized in the heart proteins to a
myocardial infarction (heart attack)
o Monoclonal antibodies can be used as therapeutic
agents
 For example, trastzumab (Herceptin) is a
monoclonal antibody useful for treating
some forms of brest cancer
 Specificity of antibodies has practical uses
o A selected antibody can be covalently attached to a resin and used in a
chromatography column
o When a mixture of proteins is added, the antibody specifically binds
its target protein and retains it on the column while the other proteins
are washed through
o The target protein can be eluted from the resin by a salt solution or
another agent
 In another technique, the antibody is attached to a radioactive label or some
other reagent that makes it easy to detect
o When the antibody binds the target protein, the label reveals the
presence of the protein in a solution or tis location in a gel or live cell
 ELISA (enzyme-linked immunosorbent assay) can be used to rapidly screen
for and quantify an antigen in a sample
o Proteins in a sample are adsorbed to an inert surface
o The surface is washed with a solution of an inexpensive nonspecific
protein to block proteins introduced in subsequent steps from
adsorbing to unoccupied sites
o The surface is then treated with a solution containing the primary
antibody – an antibody against the protein of interest
o Unbound antibody is washed away, and the surface is treated with a
solution containing a secondary antibody – antibody against the
primary antibody – linked to an enzyme that catalyzes a reaction that
forms a colored product
o After unbound secondary antibody is washed away, the substrate of
the antibody-linked enzyme is added
o Product formation is proportional to the concentration of the protein
of interest in the sample
o Indirect and sandwich ELISA
 Antibodies can be used as exquisitely specific analytic reagents
to quantify the amount of a protein or other antigen present in a
biological sample
 Indirect: used to detect the presence of antibody and is for
example the basis of the test for HIV infection
 Sandwich: used to detect antigen rather than antibody
o ELISA makes use of an enzyme such as horseradish peroxidase or
alkaline phosphatase that reacts with a colorless substrate to produce a
colored product
 The enzyme is covalently linked to a specific antibody that
recognizes a target antigen
o Rapid and convenient, ELISAs can detect less than a nanogram of a
specific protein
o Can be used to detect antigen or antibody in sample – depending on
procedure
o Requires formation of an antigen-antibody complex
o Proteins are in their native state
 Western blot is an immunoblot assay where the proteins that have been
separated by a gel electrophoresis are transferred electrophoretically to a
nitrocellulose membrane
o The membrane is blocked then successively treated with primary
antibody, secondary antibody linked to enzyme, and substrate
 o A colored precipitate forms only along the band containing the protein
of interest
o Makes it possible to find a protein in a complex mixture, the
proverbial needle in a haystack
o Proteins transferred to a membrane or sheet
o Requires formation of an antigen-antibody complex
o Proteins are denatured
 Immunoblotting allows the detection of a minor component in a sample and
provides an approximation of its molecular weight
 Co-immunoprecipitation
o With a monoclonal antibody against a specific protein available, it is
also possible to determine the binding partners of that protein under a
specific set of conditions
o The sample of interest – an extract prepared from cultured cells or
isolates tissue, is incubated with the specific antibody
 Fluorescence microscopy
o Fluorescent markers provide a powerful means of examining proteins
in their biological context
o Cells can be stained with fluorescent-labeled antibodies and examined
by fluorescence microscopy to reveal the location of a protein of
interest
 X-ray crystallography
o X-rays provide the best resolution for the determination of molecular
structures because their wavelength approximately corresponds to the
length of a covalent bond
o Protein must be crystallized
o Slowly adding ammonium sulfate or another salt to a concentrated
solution of protein to reduce its solubility favors the formation of
highly ordered crystals
o Proteins frequently crystallize in their biologically active
configuration – enzyme crystals may display catalytic activity if the
crystals are suffused with the substrate
o When a narrow beam of x-rays is directed at the protein crystal, most
of the beam passes directly through the crystal while a small part is
scattered in various directions
 These scattered or diffracted x-rays can be detected by x-ray
film or by a solid state electronic director
  The scattering pattern provides abundant information about
protein structure
o Basic principles underlying this technique
 Electron scatter rays
 The scattered waves recombine
 The way in which the scattered waves recombine depends only
on the atomic arrangement
 NMR spectroscopy
o Able to reveal protein structure in solution
o Depends on the fact that certain atomic nuclei are intrinsically
magnetic
 Only a limited number of isotopes display this property – spin
o The different frequencies – chemical shifts – are expressed in
fractional units relative to the shifts of a standard compound, such as a
water-soluble derivative of tetramethyl silane that is added with the
sample
o Good technique to look at conformational changes in solution
o Interaction between nuclei that is proportional to the inverse sixth
power of the distance between them
 Cryo-Electron Microscopy
o Thin layer of protein solution is prepared in a fine grid then frozen
very quickly, trapping the molecules in an ensemble of orientations
o The sample is then placed in a transmission electron microscope under
vacuum conditions and exposed to an incident electron beam

5.3, Protein Interactions Modulated by chemical Energy: Actin,

Myosin, and Molecular Motors

The Major Proteins of Muscle Are Myosin and Actin

 Myosin and actin generate the contractile force of muscle
o Arranged in filaments that undergo transient interactions and slide
past each other to bring about contraction
o Actin and myosin make up more than 80% of the protein mass muscle
 Myosin
o Six subunits
 Two heavy chains and two light chains
 Heavy chains account for much more of overall structure
 o At their carboxyl termini, they are arranged as
extended a-helices wrapped around each other in a
fibrous, left-handed coiled coil similar to that of a-
keratin
o At the amino terminus, each heavy chain has a
large globular domain containing a site where ATP
is hydrolyzed
 The light chains are associated with the globular domains
o When myosin is treated briefly with the protease trypsin, much of the
fibrous tail is cleaved off, dividing the protein into components called
light and heavy meromyosin
o The globular domain – called myosin sub fragment 1, or S1, or simply
myosin head group – is liberated from heavy meromyosin by cleavage
with papain – leaving myosin sub fragment 2 or S2
 The S1 fragments can be crystallized and their overall structure
o In muscle cells, molecules of myosin aggregate to form structures
called thick filaments
 These rod like structures are the core of the contractile unit
 Within a thick filament, several hundred myosin molecules are
arranged with their fibrous tails associated to form a long
bipolar structure
 The globular domains project from either end of this structure,
in regular stacked arrays
 Actin
o Abundant in almost all eukaryotic cells
o Molecules of monomeric actin, G-actin, associate to form a long
polymer called F-actin
o The thin filament consists of F-actin and the proteins troponin and
tropomyosin
 The filamentous parts of thin filaments assemble as successive
monomeric actin molecules add to one end
 On addition, each monomer binds ATP, then hydrolyzes it to
ADP, so every actin molecule in the filament is complexed to
ADP
o This ATP hydrolysis by actin functions only in the assembly of the
filaments; it does not contribute directly to the energy expended in
muscle contraction
 Each actin monomer in the thin filament can bind tightly and
specifically to one myosin head group
Additional Proteins Organize The Thick and Thin Filaments Into Ordered
Structures
 Skeletal muscle consists of parallel bundles of muscle fibers
o Each fiber a single, large, multinucleated cell, formed from many
cells fused together – a single fiber often spans the length of the
muscle
o Each fiber contains about 1,000 myofibrils, each with a vast number
of regularly arrayed thick and thin filaments complexed to other
proteins
 A system of flat, membranous vesicles called the sarcoplasmic reticulum
surrounds each myofibril
 Muscle fibers reveal alternating regions of high and low electron density,
called the A and I bands
o These bands arise from the arrangement of thick and thin filaments,
which are aligned and partially overlapping
o The I band is the region of the bundle that in cross section would only
contain thin filaments
o The darker A band stretches the length of the thick filament and
includes the region where parallel thick and thin filaments overlap
 Z disk: bisects the I band, perpendicular to the thin filaments and serving as
an anchor to which the thin filaments are attached
 M line: a thin line that bisects the A band, a region of high electron density
in the middle of the thick filaments
 Sarcomere: the entire contractile unit consisting of bundles of thick
filaments interleaved at either end with bundles of thin filaments
o The arrangement of interleaved bundles allows the thick and thin
filaments to slide past each other, causing a progressive shortening of
each sarcomere
 The thin actin filaments are attached at one end to the Z disk in a regular
pattern
o This assembly includes the minor muscle proteins a-actin, desmin,
and vimentin
o Thin filaments also contain a protein called nebulin – thought to be
structured as an a-helix that is long enough to span the length of the
filament
o the M line similarly organizes the thick filaments
 it contains the proteins paramysoin, C-protein, and M-protein
  The titins are another class of proteins that are the largest single polypeptide
chains discovered thus far
o They link the thick filaments to the Z disk, providing additional
organization to the overall structure
 Nebulin and titin are believed to act as “molecular rulers” – regulating the
length of the thin and thick filaments, respectively
o Titin extends from the Z disk to the M line – regulating the length of
the sarcomere and preventing overextension of the muscle
o The characteristic sarcomere length varies from one muscle tissue to
the next in a vertebrate, largely due to the different titin variants in
the tissues

Myosin Thick Filaments Slide Along Actin Thin Filaments

 The interaction between actin and myosin involves weak bonds
o When ATP is not bound to myosin, a face on the myosin head group
tightly binds to actin
o When ATP binds to myosin and is hydrolyzed to ADP and P, a
coordinated and cyclic series of conformational changes occurs in
which the myosin releases the F-actin subunit and binds another
subunit farther along the thin filament
 The cycle has four major steps
o 1. ATP binds to myosin and a cleft in the myosin molecule opens,
disrupting the actin-myosin interaction so that the bound actin is
released (ATP is hydrolyzed in this step)
o 2. There is a conformational change in the protein to a “high energy”
state that moves the myosin head and changes its orientation in
relation to the actin thin filament
 Myosin then binds weakly to an F-actin subunit closer to the Z
disk than the one just released
o 3. Another conformational change occurs in which the myosin cleft
closes, strengthening the myosin-acting binding
o 4. A “power stroke” during which the conformation of the myosin
head returns to the original resting state, its orientation relative to the
bound actin changing so as to pull thetail of the myosin toward th Z
disk
 ADP is then released to complete the cycle, each cycle
generates about 3 to 4pN of force and moves the thick filament
5 to 10 nm relative to the thin filament
  Because there are many myosin heads in a thick filament, some are bound to
thin filaments at any given moment
o This prevents thick filaments from slipping backward when an
individual myosin head releases the actin subunit to which it was
bound
o The thick filament actively slides forward past the adjacent thin
filaments
o This process, coordinated among the many sarcomeres in a muscle
fiber, brings about the muscle contraction
 This interaction between the actin and myosin must be regulated so that
contraction occurs only in response to appropriate signals from the nervous
system
o The regulation is mediated by a complex of two proteins: tropomyosin
and troponin
 Tropomyosin binds to the thin filament, blocking the
attachment sites for the myosin head groups
 Troponin is a Ca2+ binding protein
 A nerve impulse causes release of Ca2+ ions from the
sarcoplasmic reticulum
 The released Ca2+ binds to troponin and causes a
conformational change in the tropomyosin-troponin complexes
 Exposing the myosin-binding sites on the thin filaments
 Contraction follows
 Working skeletal muscle requires two types of molecular functions that are
common in proteins – binding and catalysis
o The actin-myosin interaction, a protein-ligand interaction like that of
immunoglobulins with antigens, is reversible and leaves the
participants unchanged
o When ATP binds myosin, however, it is hydrolyzed to ADP and Pi
 Myosin is not only an actin-binding protein, it is also an ATPase – an
enzyme

REVIEW

1. As the partial pressure of carbon dioxide increases, the affinity of oxygen

binding to hemoglobin
a. Decreases
b. Increases
c. Stays the same
 d. Increases then decreases
e. Decreases then increases
2. What is the functional role of the “distal histidine”
a. It donates a hydrogen bond to the bound oxygen molecule to
stabilize the complex between iron and oxygen
b. It improves access of carbon monoxide to the heme and inhibits
oxidation of iron to the ferric state
c. It binds tightly to the heme ion to stabilize the complex between iron
and oxygen
d. It stabilizes the oxygen complex and catalyzes the oxidation of iron to
the ferric state
e. It stabilizes the complex between Fe2+ and dioxygen
3. Consider the oxygen-binding profile at three different pH values – 7.6, 7.4,
and 7.2 – which statement is MOST accurate? (lower pH  higher
concentration of hydrogen ions  lower affinity) (lowest affinity for oxygen
has highest Kd value – bottom curve will have the lowest pH)
a. Curve X most likely corresponds to pH 7.2
b. Curve Z most likely corresponds to pH 7.6
c. Hb has a higher affinity for oxygen at the pH of curve Z
d. Curve Y most likely corresponds to pH 7.4
e. pH has no effect on oxygenation of hemoglobin
4. Which statement about antibodies is INCORRECT?
a. Antibodies bind to antigens via induced fit
b. Antibodies typically bind to antigens more strongly than enzymes
bind to substrates
c. Antibodies can be both soluble and membrane-bound
d. Antibodies never release antigens once they are bound together
e. More than 100 million different antibodies can be produced in humans

5. Antigens typically bind to antibodies via which pair of interactions

a. Covalent bonding
b. Hydrophobic interactions
c. Hydrogen bonding
d. Both covalent bonding and hydrophobic interactions
e. Both hydrophobic interactions and hydrogen bonding
6. You want to purify a specific soluble protein from a solution of lysed cells
so that you can conduct biochemical test of the protein’s function – to best
achieve this, which type of antibody would be MOST effective to use in
your affinity purification?
a. Monoclonal IgE
 b. Polyclonal IgE
c. Monoclonal IgG
d. Polyclonal IgG
e. Monoclonal IgM
7. Which statement about antigen-antibody interactions is CORRECT?
a. Antigens bind to antibodies cooperatively
b. Antigens bind to antibodies through induced fit
c. Antigens bind to antibodies at both the Fab and fc parts of the
antibody
d. Molecules with molecular weights of less than 5,000 Daltons are
exceptionally antigenic and bind to antibodies extremely well
e. Antigens bind only to antibodies that have a performed binding site
that is perfectly complementary to the antigen
8. Macrophages bind to the _________ region of IgG antibodies, thereby
triggering phagocytosis of antibody-antigen complexes.
a. Fab
b. Fc
c. Epitope
d. Hinge
e. Antigen
9. Expert thinking question: According to the data, what is the approximate
monomeric molecular weight of sPLA2? Why is it biologically relevant that
sPLA2 required high acetonitrile concentration to elute off the column?
10. Which type of covalent bonds hold antibody structures together?
a. Hydrogen bonds
b. Disulfide bonds
c. Van der Waals interactions
d. London forces
e. Ionic bonds
11.What statement is CORRECT concerning the oxygenation plot of proteins X
and Y shown below? (protein Y has a higher Kd value; fetal hemoglobin has
a higher affinity for oxygen than adult hemoglobin so lower Kd value, graph
is sigmoidal so not myoglobin or single binding site because it would be
hyperbolic))
a. Protein Y exhibits tighter oxygen binding than protein X
b. Protein Y corresponds to fetal hemoglobin, and protein X corresponds
to normal adult hemoglobin
c. Protein X corresponds to fetal hemoglobin, and protein Y
corresponds to normal adult hemoglobin (X has the higher binding
curve, Y is the lower)
 d. Protein X corresponds to myoglobin, and protein Y corresponds to
hemoglobin
e. Proteins X and Y contain a single binding site
12.What type of binding is indicated by a sigmoidal-shaped binding curve?
a. Competitive
b. Noncompetitive
c. Uncooperative
d. Cooperative
e. Reversible
13.Choose the protein described by the following Hill plot: n=1 (remember n=1
means no cooperation, n above 1 = positive cooperation, n below 1 =
negative cooperation)
a. Adult hemoglobin
b. Fetal hemoglobin
c. Myoglobin
d. Allosteric enzyme
e. Globin with multiple binging sites
14.Sickle-cell anemia results in:
a. Decreased production of a chains in hemoglobin
b. Increases production of B chains in hemoglobin
c. Loss of the heme group in mutated globins
d. Increases solubility of deoxyhemoglobin
e. Formation of large aggregates of the mutated globins
15.What is NOT true of the following equilibrium: CO2 + H2O  H2CO3
a. An increase in the pressure of CO2 will result in a decrease of pH
b. This reaction is catalyzed by carbonic anhydrase
c. The H2CO3 dissociates to H+ and bicarbonate ion, HCO3-
d. The majority of CO2 is transported to the lungs in the form of HCO3-
e. This reaction does not need the catalyst
16.What is the functional role of 2,3-BPG?
a. Stabilization of the t state of hemoglobin
b. Stabilization of the R state of hemoglobin
c. Covalent modification of the R state of hemoglobin
d. Covalent modification of the T state of hemoglobin
e. Saving the equilibrium between the R and T states of hemoglobin
17.Select all that apply. The fetal hemoglobin has:
a. A lower binding affinity for 2,3-BPG than adult hemoglobin
b. A lower binding affinity for oxygen than adult hemoglobin
c. A higher binding affinity for 2,3-BPG than neuroglobin
d. A higher binding affinity for oxygen than adult hemoglobin
e. The same level of binding affinity for oxygen