Biochemistry Exam 2

T-State

"Tense"; The T-state is the deoxy form of hemoglobin (meaning that it lacks an oxygen species) and is also known as "deoxyhemoglobin". ... The sequential model of cooperativity model maintains that when one strand of hemoglobin binds oxygen, the hemoglobin rearranges in a manner that favors additional oxygen binding.

R-State

"relaxed"; The R-state is the fully oxygenated form: "oxyhemoglobin." -For hemoglobin to function efficiently, the T state must remain stable until the binding of sufficient oxygen has converted it into the R state. However, the T state of hemoglobin is highly unstable, pushing the equilibrium so far toward the R state that little oxygen would be released in physiological conditions. Thus, an additional mechanism is needed to properly stabilize the T state.

Antibody

(also called an immunoglobulin, Ig) is itself a protein (Figure 3.17); it is synthesized by vertebrates in response to the presence of a foreign substance, called an antigen. Antibodies have specific and high affinity for the antigens that elicited their synthesis. The binding of antibody to antigen is a step in the immune response that protects the animal from infection

What does pO2 refer to in a solution like blood? In the blood, is there some O2 bound and some unbound? What form of oxygen does the pO2 refer to?

- Reflects the amount of oxygen gas dissolved in the blood. Measures the effectiveness of lungs pulling O2 from environment - Unbound and bound O2 - Unbound O2

Describe the process of western blotting. What is it used for? How is it similar to and different from affinity chromatography?

1. Transfer 2. Blocking 3. Primary antibody incubation 4. Secondary antibody incubation 5. Protein detection 6. Western blotting analysis Western blotting detects a specific protein in a blood or tissue sample. Uses gel electrophoresis to separate the sample's proteins and then transferred out of the gel to the surface of a membrane. Similarities: works on binding interactions - Western has binding of antibodies and unbinding, affinity has binding by antibody/antigen or enzyme/substrate or enzyme/inhibitor Differences: Western blot - detection of a specific protein from mixture, Affinity chromatography - purification of a protein based on specific binding interactions

2,3-Bisphosphoglycerate in Red Cells is Crucial in Determining the Oxygen Affinity of Hemoglobin

2, 3-Bisphosphoglycerate (2,3-BPG) stabilizes the T state of hemoglobin and thus facilitates the release of oxygen. 2, 3-BPG binds to a pocket in the hemoglobin tetramer that exists only when hemoglobin is in the T state.

How many oxygens can one molecule of hemoglobin bind? How many oxygens can one subunit of hemoglobin bind?

4 oxygens can be bound to one molecule of hemoglobin. 1 oxygen can bind per subunit of hemoglobin.

Hemoglobin subunits

4. alpha and beta subunits. a1b1-a2b2

Daniella prepares a 1 mg/ml1 mg/ml myoglobin solution. The molecular weight of myoglobin is 17.8 kDa.17.8 kDa. Given that the 𝜖ϵ of myoglobin is 15,000 M−1cm−1,15,000 M−1cm−1, calculate the absorbance of the myoglobin solution across a 1 cm1 cm path. Calculate your answer to two decimal places. What percentage of the incident light is transmitted by this solution? Calculate your answer to one decimal place. transmitted percentage =

A = 0.84 Transmitted percentage = 14.4%

Absorbance of a solution formula

A = log10 (Io/I) Io = incident-light intensity I = transmitted-light intensity A = ecl e = molar absorption coefficient (extinction coefficient) in units of M−1cm−1,M−1cm−1 c = molar concentration l = path length in centimeters

Complete the passage about matrix‑assisted laser desorption/ionization time‑of‑flight, or MALDI‑TOF, mass spectrometry.

A MALDI‑TOF mass spectrometer uses a laser beam to vaporize the protein sample, which is embedded in a volatile matrix. Collisions between the gaseous protein molecules ionize the sample. An electrostatic potential accelerates the ions through the flight tube toward the detector, and the lightest ions arrive at the detector first.

Fluorescence microscopy

A microscope equipped to examine material that fluoresces under ultraviolet light. Fluorescence microscopy is based on the principle that fluorescent materials emit visible light when they are irradiated with ultraviolet rays or with violet-blue visible rays.

Hemoglobin

A red blood cell protein that transports oxygen from the lungs to the tissues. An allosteric protein that displays cooperativity in oxygen binding and release.

Monoclonal antibodies

A type of protein that is made in the laboratory and can bind to certain targets in the body, such as antigens on the surface of cancer cells.

How would you separate two proteins that have similar physical characteristics (e.g., size and pI), but have very different functional characteristics?

Affinity chromatography. Separates compounds based on their different weak affinities to an immobilized target

How does the oxygen binding affinity for the "new" hemoglobin depicted in the right shifted curve compare with the original hemoglobin?

Affinity decreases. More O2 needed to achieve the same saturation

Hemoglobin binds oxygen cooperatively

An oxygen binding curve is a plot of the fractional saturation versus the oxygen concentration, which is shown as partial pressure (in units of torr). - Torr is a unit of pressure equal to that exerted by a column of mercury 1 mm high at 0°C and standard gravity (1 mm Hg). Myoglobin displays a hyperbolic oxygen binding curve, while hemoglobin exhibits a sigmoid curve, indicating that O2 binding and release is cooperative. The cooperativity allows hemoglobin to bind oxygen in the lungs, where it is plentiful, and release oxygen at the tissues, where it is scarce.

Mass spectrometry

Analytical technique. Quantify known materials, identify unknown compounds within a sample, and to elucidate the structure and chemical properties of different molecules

How does YO2 change with increasing pO2 levels and use the term oxygen binding in your answer.

As pO2 increases, oxygen binding will increase as its available sites fill up. Therefore, as pO2 increases, YO2 increases. Increases amount of O2 binding

Importance of increasing salt concentration in ion exchange chromatography

As salt concentration is added, the sodium ions will compete with the positively charged groups on the protein for binding to the column, which will lead to the protein being washed away from the column and into the fraction.

How do the levels of O2 in the atmosphere compare at sea level versus high altitude? Predict the blood levels of BPG for people who live at high altitudes, like Denver. How will those levels of BPG help their hemoglobin function better?

BPG decreases hemoglobins affinity to O2 and because of that O2 is more easily delivered to the tissues. Being in higher altitudes increases the need for O2 to be delivered to the tissues since there is less O2. BPG will therefore increase for those who live at high altitudes. Hemoglobin can therefore deliver more O2 from lungs to tissues

SDS-PAGE gel electrophoresis separates peptide and proteins based on size. View the gel electrophoresis animation found on the companion website. One of the proteins in the animation contains subunits. How are these subunits associated with each other in the intact protein?

Binded by charges. Intramolecular forces. Ionic attraction binds them, detergent added to make uniformly negative, electric current added

Myoglobin

Binds oxygen in muscle cells. The binding of oxygen by myoglobin is not cooperative

Complete the passage.

Biochemists working with proteins use a diverse set of treatments to analyze protein sequences. Denaturing agents unfold proteins. Some denaturing agents are reversible, and allow the proteins to be renatured after the procedure. A strong urea solution is a common reversible denaturant. Additional treatment with β-mercaptoethanol completely denatures proteins that contain disulfide bonds. The Edman degradation method of protein sequencing labels the amino‑terminal residue of a peptide with phenyl isothiocyanate. Edman degradation is a feasible way to sequence short peptides. Biochemists can break large proteins into peptide fragments to assist in sequence determination. Chymotrypsin catalyzes hydrolysis of peptide bonds after aromatic residues. Cyanogen bromide molecules cleave peptide bonds after methionine residues. Trypsin hydrolyzes peptide bonds after lysine and arginine residues.

Which five statements about hemoglobin and myoglobin structure are true? Molecular oxygen binds irreversibly to Fe2+Fe2+ in heme. Each hemoglobin or myoglobin molecule can bind four oxygen molecules. Both hemoglobin and myoglobin contain a prosthetic group called heme, which contains a central iron atom. By itself, heme is not a good oxygen carrier. It must be part of a larger protein to prevent oxidation of the iron atom. Hemoglobin is a heterotetramer, whereas myoglobin is a monomer. Heme is composed of an organic protoporphyrin component and a metal atom. Each iron atom can form six coordination bonds. One of these bonds is formed between iron and oxygen.

Both hemoglobin and myoglobin contain a prosthetic group called heme, which contains a central iron atom. By itself, heme is not a good oxygen carrier. It must be part of a larger protein to prevent oxidation of the iron atom. Hemoglobin is a heterotetramer, whereas myoglobin is a monomer. Heme is composed of an organic protoporphyrin component and a metal atom. Each iron atom can form six coordination bonds. One of these bonds is formed between iron and oxygen.

Heme group

Consists of an organic component called protoporphyrin and a central iron ion (ferrous Fe2+ ion bc that's the only form that can bind O2)

The illustration shows several oxygen‑dissociation curves. Assume that curve 3 corresponds to hemoglobin with physiological concentrations of CO2 and 2,3-bisphosphoglycerate (2,3-BPG) at pH 7.7. Assign each perturbation or the lack of a perturbation to the curve it represents.

Curve 1: a loss of quaternary structure Curve 2: a decrease in CO2, an increase in pH Curve 3: no perturbation Curve 4: an increase in 2,3-BPG

Myoglobin and Hemoglobin binding

Dependent on the presence of a heme group

Is the central cavity of hemoglobin the same or different than the oxygen binding site of hemoglobin?

Different

Compare and contrast ELISA and western blotting (immunoblotting) by placing the phrases to the technique that they describe. If a phrase describes both ELISA and western blotting, place it under Both.

ELISA: - proteins are in their native state - can be used to detect antigen or antibody in a sample, depending on procedure Western Blot with SDS-PAGE: - proteins are denatured - proteins transferred to a membrane or sheet Both: - requires formation of an antigen-antibody complex

Upon centrifugation, particles sediment at different rates due to their physical properties. Tropomyosin is a rod-shaped protein with a mass of 70 kDa and a sedimentation coefficient of 2.6S. Hemoglobin is a spherical protein with a slightly smaller mass of 65 kDa, but a much higher sedimentation coefficient of 4.31S. Which property of tropomyosin accounts for its slow sedimentation?- - elongated shape - high mass low - partial specific volume - overall net charge - high particle density

Elongated shape

Consider a mixture of four proteins with various molecular weights. A histone molecule weighs 15 kDa, a p53 molecule weighs 53 kDa, an actin molecule weighs 42 kDa, and an IgG molecule weighs 150 kDa. Arrange the molecules in order of their elution from a gel filtration column.

Elutes first IgG (150 kDa) p53 (53 kDa) actin (42 kDa) histone (15 kDa) Elutes last

Cooperativity

Enhances Oxygen delivery

How does 2,3-BPG lower the oxygen affinity of hemoglobin so significantly?

Examination of the crystal structure of deoxyhemoglobin in the presence of 2,3-BPG reveals that a single molecule of 2,3-BPG binds in the center of the tetramer, in a pocket present only in the T form (Figure 7.17). On the T-to-R transition, this pocket collapses, and 2,3-BPG is released. Thus, in order for the structural transition from T to R to take place, the bonds between hemoglobin and 2,3-BPG must be broken. In the presence of 2,3-BPG, more oxygen-binding sites within the hemoglobin tetramer must be occupied in order to induce the T-to-R transition, and so hemoglobin remains in the lower-affinity T state until higher oxygen concentrations are reached. This mechanism of regulation is remarkable because 2,3-BPG does not in any way resemble oxygen, the molecule on which hemoglobin carries out its primary function. 2,3-BPG is referred to as an allosteric effector.

True or false. Two different enzymes may catalyze the same reaction using different mechanisms, but the rate of the reaction must be the same

False

Which molecules are bound to hemoglobin when hemoglobin is in the R state? Fe2+ CO2 oxygen 2,3‑bisphosphoglycerate Fe3+

Fe2+, oxygen

Arrange the steps to synthesize a high-density peptide array on a solid support in the correct order.

First step Determine the desired location and sequence of the peptides to synthesize. Attach amino acids with photolabile protecting groups to solid support. Shine light on selected regions of the solid support to release protecting groups. Add the next amino acid, which will react with exposed amino acids in selected regions. Last step

Hill plot

For myoglobin, the Hill plot is linear with a slope of 1. For hemoglobin, the Hill plot is not completely linear, because the equilibrium on which the Hill plot is based is not entirely correct.

How would you separate two proteins that differ greatly by size?

Gel electrophoresis. Sending electrical current through gel loaded with samples to separate by charge

If your experimental goal is to collect each intact protein for further analysis, would gel filtration or SDS-PAGE be preferable? Why?

Gel filtration because the protein can be recovered

Describe the principles involved in the separation of proteins by size exclusion "gel filtration" chromatography

Gel filtration chromatography works by inclusion/exclusion of a molecule to enter the beads on a column and works on molecular size. The size of the molecule corresponds to where it will emerge. Larger molecules will flow more rapidly through the column and its beads because large molecules are located only in the solution between the beads and therefore a smaller volume is accessible. Medium molecules will then appear as some of them can occasionally enter the beads and flow through the column to an intermediate position. Small molecules exit the end as they can enter the beads and are distributed in the solution both inside the beads and around them.

Studies of oxygen transport in pregnant mammals show that the O2O2‑saturation curves of fetal (HbF) and maternal (HbA) hemoglobin are markedly different when measured under the same conditions. Which hemoglobin has a higher affinity for O2O2 at the tissue pO2pO2 of around 4 kPa? HbA HbF cannot be determined from this data The allosteric inhibitory effector BPG binds to both the A form and the F form. Both forms show a lower affinity for O2O2 in the presence of BPG. However, the inhibitory effect of BPG is larger for HbA. The different O2O2 affinity indicated by the curves has been attributed to different BPG affinity for the two Hb forms. Complete the statement. BPG binds tighter to HbA , which lowers its affinity for O2O2 . Which statement that best explains the role of BPG in O2O2 transport from mother to fetus? HbA‑bound O2O2 will tend to move to HbF because HbA binds an additional BPG per tetramer when it enters placental circulation. HbF will extract O2O2 from oxygenated HbA because BPG enhances O2O2 binding to HbF. In the placental circulation, HbF will load up on O2O2 as BPG dissociates from HbA and binds to HbF. HbA‑bound O2O2 will tend to move to HbF because HbF has a lower affinity for BPG, an allosteric inhibitor of O2O2 binding.

HbF HbA, lowers HbA‑bound O2O2 will tend to move to HbF because HbF has a lower affinity for BPG, an allosteric inhibitor of O2O2 binding.

Hemoglobin Assembly

Hemoglobin consists of four chains: two identical α chains and two identical β chains. Many of the helices in each subunit are arranged in a pattern also found in myoglobin, a structure called the globin fold.

A and B chains

Hemoglobin consists of four polypeptide chains, two identical α chains, and two identical β chains (Figure 7.6). Each of the subunits consists of a set of α helices in the same arrangement as the α helices in myoglobin

Hemoglobin cooperativity

Hemoglobin cooperativity incorporates aspects of both the concerted and sequential models of allostery. - It is concerted in that in hemoglobin with three O2 bound, the remaining subunit is in the R state. - It is sequential in that in hemoglobin with one O2 bound, the remaining subunits are in the T state.

Hemoglobin affinity

Hemoglobin has a greater affinity for oxygen when the partial pressure of oxygen is high and has a lower affinity when the partial pressure of oxygen is low

Determine which statements apply to hemoglobin, myoglobin, or neither.

Hemoglobin: The oxygen dissociation curve is sigmoidal in shape ("S"‑shaped). As oxygen binds to this molecule the shape of the molecule changes, enhancing further oxygen binding. The binding pattern for this molecule is considered cooperative. This molecule delivers oxygen more efficiently to tissues. Myoglobin: The oxygen dissociation curve is hyperbolic in shape. This molecule has a greater affinity for oxygen. Neither: Oxygen binds irreversibly to this molecule. Carbon monoxide binds at an allosteric site, lowering oxygen binding affinity.

The pKa of an acid depends partly on its environment. Predict the effect of each of the following environmental changes on the pKa of a glutamic acid side chain.

If a lysine side chain is brought into proximity, the pKa of the glutamic acid chain is lowered. If the terminal carboxyl group of the protein is brought into proximity to the glutamic acid side chain, the pKa of the side chain is raised. If the glutamic acid side chain is shifted from the outside of the protein to a nonpolar site inside of the protein, the pKa is raised.

Compare the types of information we obtain about proteins using molecular exclusion chromatography and SDS-PAGE

In molecular exclusion chromatography we obtain the molecular weight of the proteins in the sample and their relative size. In SDS-PAGE we obtain subunit sizes, molecular weight, and electrical charge of proteins - a specific size.

Proximal histidine

In myoglobin, the fifth coordination site is occupied by the imidazole ring of a histidine residue from the protein. This histidine is referred to as the proximal histidine.

Cryo-electron microscopy

In order to perform cryo-EM, a thin layer of the protein solution is prepared in a fine grid then frozen very quickly, trapping the molecules in an ensemble of orientations. The sample is then placed in a transmission electron microscope under vacuum conditions and exposed to an incident electron beam. Each protein interacts with the beam to produce a two-dimensional projection on the image capture device, or detector. Many projections are detected, each capturing a molecule in a different orientation (Figure 3.49A). Using a process called single-particle analysis, computers use these projections to build a three-dimensional representation of the protein (Figure 3.49B).

Distal histidine

In particular, the binding pocket of myoglobin includes an additional histidine residue (termed the distal histidine) that donates a hydrogen bond to the bound oxygen molecule

Heme group binding

Iron in the middle of protoporphyrin bound to four nitrogens, iron can form two additional bonds at the fifth and sixth coordination sites. Fifth coordination site is occupied by an imidazole ring of a histidine called the proximal histidine. Sixth coordination site binds oxygen. Upon oxygen binding, the iron moves into the plane of the protoporphyrin ring.

Two-dimensional electrophoresis

Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution separations by two-dimensional electrophoresis. A single sample is first subjected to isoelectric focusing. This single-lane gel is then placed horizontally on top of an SDS-polyacrylamide slab. The proteins are thus spread across the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing. They then undergo electrophoresis again in a perpendicular direction (vertically) to yield a two-dimensional pattern of spots. In such a gel, proteins have been separated in the horizontal direction on the basis of isoelectric point and in the vertical direction on the basis of mass.

How would you separate two proteins that differ by pI?

Isoelectric focusing. Applying an electric field to protein within a pH gradient. Proteins separate as they migrate through the pH gradient in response to the applied voltage

Match the characteristics of the two steps of two‑dimensional electrophoresis.

Isoelectric focusing: - proteins are separated by charge - carried out in a pH gradient - final net protein charge is zero SDS-PAGE: - proteins are separated by mass - carried out in a detergent solution - final net protein charge is negative

Make a generalization about hemoglobin structure as it relates to hemoglobin function

Low affinity to high affinity Tense state - deoxygenated Relaxes state - oxygenated

A researcher resolves a mixture of peptides using isoelectric focusing. Order the peptides based on their relative positions in the immobilized pH gradient strip at the end of the experiment. Arg‑Ala‑Lys‑Lys Arg‑Gly‑Glu‑Lys Arg‑Leu‑Ala‑Arg Asp‑Ala‑Leu‑Asp Glu‑Gly‑Glu‑Asp

Low pH Glu‑Gly‑Glu‑Asp Asp‑Ala‑Leu‑Asp Arg‑Gly‑Glu‑Lys Arg‑Leu‑Ala‑Arg Arg‑Ala‑Lys‑Lys High pH

Oxygen binding measurement

Measured as a function of the partial pressure of oxygen

Salting out

Most proteins are less soluble at high salt concentrations, an effect called salting out. The salt concentration at which a protein precipitates differs from one protein to another. Hence, salting out can be used to fractionate proteins.

Classify each characteristic as describing myoglobin or hemoglobin. One or more characteristics may not be used.

Myoglobin: monomeric completely noncooperative n=1 Hemoglobin: tetrameric partially cooperative n>1 sigmoidal oxygen‑binding curve

Distinguish between native gel electrophoresis of proteins and SDS-PAGE of proteins in terms of the source of charge on the proteins being separated

Native gel electrophoresis of proteins utilizes a non-denaturing gel so that molecules can be separated based on their size, charge, and shape. In native gel electrophoresis the proteins will have their native charge. In SDS PAGE electrophoresis there is a denaturing gel and molecules are separated based on their molecular weight. The SDS in SDS PAGE will denature the proteins by breaking the disulfide bonds and therefore give the proteins negative charges.

Consider the mass spectrum. Determine the sequence of the peptide from the mass spectrum. Notice that two of the residues, L and R, are already identified for you. Assume that any residues with mass around 113 are leucine. Enter your answer using one‑letter amino acid abbreviations, without dashes.

N‑terminal FEDLQGLGELR C‑terminal

Gel Filtration Chromatography

Preparative technique. Separates specific samples by size

Ion exchange chromatography

Preparative technique. Separation and determination of ionic compounds

Affinity chromatography

Preparative technique. Separation on a specific binding interaction between an immobilized ligand and its binding partner

Consider an experiment where the researcher used Western blotting to detect protein X, which weighs 50 kDa in the whole cell lysates of normal cells and cancer cells. Why are there two bands present in one of the lanes? Protein X is cleaved in cancer cells. Protein X forms a dimer in cancer cells. Protein X is post‑translationally modified in cancer cells. The primary antibody is not specific to protein X.

Protein X is post-transitionally modified in cancer cells

Describe the principles involved in protein purification by affinity chromatography. Be thorough.

Protein purification via affinity chromatography via binding, washing, and eluting. In the binding step, the targeted protein is covalently bonded to a group that is recognized by that specific protein. In the washing step, a mixture of proteins is added to the column and washed with buffer to remove unbound proteins. In the eluting step, the targeted protein is released from the column by the addition of a high concentration of the group recognized to the targeted protein or by altering the conditions in which the binding affinity can be decreased. Therefore, the targeted proteins will end up in the flow through.

Select the true statements about SDS‑PAGE, a method of separating proteins. Assume that SDS‑PAGE is performed under reducing conditions. Protein‑SDS complexes migrate toward the positive electrode. SDS‑PAGE utilizes agarose gel to separate proteins. Proteins are visualized using a dye that binds to the gel matrix, but not to proteins. Smaller proteins migrate faster through the polyacrylamide gel. Sodium dodecyl sulfate binds proteins, resulting in protein‑SDS complexes that are similar in size. Protein‑SDS complexes have similar mass to charge ratios; therefore, separation is by size.

Protein-SDS complexes migrate toward the positive electrode, smaller proteins migrate faster through the polyacrylamide gel, protein-SDS complexes have similar mass to charge ratios; therefore, separation is by size

Dialysis

Proteins can be separated from small molecules such as salt by dialysis through a semipermeable membrane, such as a cellulose membrane with pores. The protein mixture is placed inside the dialysis bag, which is then submerged in a buffer solution that is devoid of the small molecules to be separated away.

Cation exchange

Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density. This procedure is also referred to as cation exchange to indicate that positively charged groups will bind to the anionic beads.

Why does YO2 ranges from 0 to 1. What does your group think YO2 = 0.5 means at the molecular level for a sample of hemoglobin molecules?

Ratio of available sites of oxygen binding. 50% of binding sites are filled

Explain how SDS-PAGE produces separation of proteins on the basis of subunit size

SDS-PAGE breaks apart the subunits of the protein by breaking the disulfide bonds which then allows the protein to be separated by subunit size. SDS-polyacrylamide gel electrophoresis produces separation of proteins on the basis of subunit size because the charge of the molecules is proportional to the size. The larger the subunit, the larger their negative charge. Smaller subunits will move quicker through the gel and will be observable closer to the positive pole of the gel while the larger subunits will be immobile and remain closer to the top of the gel.

Gel filtration (size exclusion) chromatography is another technique used to separate proteins based on size. View the gel filtration animation found on the companion website. Imagine you have a mixture of proteins. If your experimental goal is to determine as accurately as possible the molecular weight of proteins in the mixture, would gel filtration or SDS-PAGE be preferable? Why?

SDS-PAGE: specific size Gel filtration: relative size

The graph represents the adult hemoglobin binding curve (in green) at pH 7.4 in the presence of 2,3‑bisphosphoglycerate. The hemoglobin binding curve has a sigmoidal shape, due to four interacting oxygen‑bound sites. For comparison, the myglobin binding curve has only one oxygen‑bound site and has a hyperbolic curve. For each of the six scenarios, determine whether the hemoglobin binding curve would shift left or shift right.

Shifts left The adult hemoglobin (HbA) is replaced by an infant's fetal hemoglobin (HbF). Hemoglobin is isolated from red blood cells and stripped of 2,3‑bisphosphoglycerate. Tetrameric hemoglobin is dissociated into its subunits. Shifts right The blood pH drops from 7.4 to 7.2. The CO2CO2 concentration in the blood increases. The concentration of 2,3‑bisphosphoglycerate increases during acclimation to high altitude.

Explain how size exclusion chromatography could be used to estimate the molecular weight of intact proteins

Size exclusion chromatography can be used to estimate the molecular weight of intact proteins because the size of the of the molecules will determine its ability to enter the beads. The molecular weight of intact proteins can be determined by how fast the molecules are eluted into the fractions. Larger molecules will therefore not enter the porous beads and exit faster through the column since a smaller volume of the column is accessible to them. Medium molecules will exit the column second because they can sometimes enter the beads and small molecules will exit the column last because they interact with the solution inside and between the beads. From here, molecular weight can be estimated by observing the flow through.

Each of the given statements describes a type of column chromatography. Match the statements to the type of chromatography they describe. If a statement can describe all of the types, place that statement in the All category. (Note that size‑exclusion chromatography may also be called gel filtration or molecular‑exclusion chromatography.)

Size-exclusion chromatography (gel filtration): - separates molecules by size - the stationary phase contains cross-linked polymers with different pore sizes Affinity chromatography: - can separate molecules based on protein-ligand binding - the stationary phase has a covalently bound group to which a protein in the mobile phase can bind Ion-exchange chromatography: - separates molecules by size - the stationary phase may contain negatively or positively charged groups All: - use a mobile phase and a stationary phase to separate proteins

Size-exclusion column chromatography and SDS-PAGE are methods used to separate mixtures of proteins. Match each statement with the appropriate category.

Size-exclusion column chromatography: - proteins are separated in their native state - larger proteins travel faster during separation - proteins of similar molecular weight but different shape can be separated SDS-PAGE: - proteins are separated in their denatured state - smaller proteins travel faster during separation - proteins move toward the positive electrode Both: - proteins are separated by size

The figure shows a two‑dimensional gel of a mixture of proteins A, B, and C. The vertical axis represents SDS‑PAGE separation, run from top to bottom. The horizontal axis represents isoelectric focusing with a pH gradient that runs from left(high pH) to right (low pH). Proteins A, B, and C have isoelectric points of 8.2, 6.7, and 5.2, respectively. Place proteins A, B, and C in order of increasing elution volume on a size exclusion column. Assume they are monomers under the conditions of separation.

Smallest Elution Volume B A C Largest Elution Volume

Sigmoid

Such curves are referred to as sigmoid because of their S-like shape. In addition, oxygen binding for hemoglobin (p50=26 torr) is significantly weaker than that for myoglobin; A sigmoid binding curve indicates that a protein exhibits a special binding behavior. For hemoglobin, this shape suggests that the binding of oxygen at one site within the hemoglobin tetramer increases the likelihood that oxygen binds at the remaining unoccupied sites. Conversely, the unloading of oxygen at one heme facilitates the unloading of oxygen at the others. This sort of binding behavior is referred to as cooperative, because the binding reactions at individual sites in each hemoglobin molecule are not independent of one another.

Heme

The ability of myoglobin and hemoglobin to bind oxygen depends on the presence of a heme molecule. As we shall discuss in Chapter 9, heme is one example of a prosthetic group—a molecule that binds tightly to a protein and is essential for its function; The heme group gives muscle and blood their distinctive red color. It consists of an organic component and a central iron atom.

functional magnetic resonance imaging (fMRI)

The change in electronic structure that occurs when the iron ion moves into the plane of the porphyrin is paralleled by alterations in the magnetic properties of hemoglobin; these changes are the basis for functional magnetic resonance imaging (fMRI)

What features of protein structure allows for their separation by ion exchange chromatography?

The charge of the protein allows for their separation by ion exchange chromatography because the charged sample can bind to the column resin via the ionic interactions and then as salt as increased the sample will be purified and ready to recollect in the fractions.

Why is it important to dialyze a protein sample obtained by ammonium sulfate precipitation before subjecting it to ion exchange chromatography?

The dialysis of the protein sample allows for the removal of the salt or other small molecules that have effectively passed through the membrane and into the fraction. The solution is very concentrated after ammonium sulfate precipitation and need to be removed so that the protein is remaining before ion exchange chromatography. Dialysis reduces the salt that is entering the ion exchange column and increases the sample's volume.

ELISA

The enzyme-linked immunosorbent assay (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. If the antigen is present, the antibody-enzyme complex will bind to it and, in addition to the substrate, the enzyme will catalyze the reaction, generating the colored product. Thus, the presence of the colored product indicates the presence of the antigen

Consider an experiment where your goal is to isolate Bruton's tyrosine kinase (BTK) enzyme from a whole‑cell lysate. You have an affinity chromatography column with a tyrosine kinase inhibitor molecule covalently attached to the beads. The tyrosine kinase inhibitor binds and inhibits BTK. As a result of the experiment, you are able to elute BTK from the column, but in a mixture of other tyrosine kinases. Why are tyrosine kinases other than BTK present in the eluate? BTK is rapidly degraded during cell lysis. The kinase inhibitor has low specificity. The kinase inhibitor has low binding affinity. BTK is inactive in the cell.

The kinase inhibitor has low specificity

Isoelectric Focusing

The method of separating proteins according to their isoelectric point is called isoelectric focusing. The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes (small multicharged polymers) having many different pI values to electrophoresis. Isoelectric focusing can readily resolve proteins that differ in pI by as little as 0.01, which means that proteins differing by one net charge can be separated

What features of protein structure allows for their separation by gel filtration chromatography?

The molecular size of the protein structures allows for their separation by gel-filtration chromatography because smaller molecules will enter the beads on the column where they can be distributed to the aqueous solution and be discriminated from other molecules.

Fourier transform

The next step is to reconstruct an image of the protein from the observed reflections. In light microscopy or electron microscopy, the diffracted beams are focused by lenses to directly form an image. However, appropriate lenses for focusing x-rays do not exist. Instead, the image is formed by applying a mathematical relation called a Fourier transform to the measured amplitudes and calculated phases of every observed reflection. The image obtained is referred to as the electron-density map

Protoporphyrin

The organic component is made up of four pyrrole rings linked by methine bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached to the central tetrapyrrole.

What is the pH of blood in the tissue and in the lungs and why does that difference in pH contribute to hemoglobin delivering O2?

The pH of blood in the lungs is 7.6 and the pH of blood in the tissues is 7.2. Blood near the lungs has less CO2 and therefore a higher pH. The higher pH increases the affinity of hemoglobin for O2 through the Bohr effect so that hemoglobin picks up O2 in the blood from the lungs to deliver to the tissues. Because there is greater CO2 and a lower pH in the tissues, hemoglobin is forced to drop off O2 in the tissues. This is shown in the equilibrium equation by there being more H+ ions in the tissue, pH lower (measurement of H+) and O2 is being delivered. Oxygen is picked up from lungs with the hemoglobin as shown on the left

Importance of the pH of the buffer in ion exchange chromatography

The pH of the buffer is important because at certain pH's the protein and its corresponding charge will bind to the column with carboxylate groups or not. If a protein has a positive charge at pH 7, it will bind with the carboxylate groups. If the protein has a negative charge at pH 7, it will not be able to bind with the carboxylate groups. The pH also determines the protonation of the protein because increasing or decreasing the pH can create a more positively or more negatively charged and therefore can affect the ability of the protein to bind to the column. Overall, the pH affects the binding ability of the protein to the column and the protein's net charge.

Cooperative binding

The positive change in hemoglobin saturation, YO2, as more O2 is present

Complete the sentences about heme.

The prosthetic group of hemoglobin and myoglobin is heme. The organic ring component of heme is porphyrin. Under normal conditions, the central atom of heme is Fe2+. In deoxyhemoglobin, the central iron atom is displaced 0.4 Å out of the plane of the porphyrin ring system. The central atom has six bonds: four to nitrogen atoms in the porphyrin, one to a histidine residue, and one to oxygen.

Oxygen Binding Markedly Changes the Quaternary Structure of Hemoglobin

The quaternary structure of deoxyhemoglobin is referred to as the T state "tense", while that of oxyhemoglobin is the R state "relaxed". The hemoglobin tetramer can be thought of as two αβ dimers. The α1β1 dimer rotates 15 degrees relative to the α2β2 dimer on oxygen binding. This structure alteration, conversion from the T state to the R state, facilitates oxygen binding.

Globin fold

The recurring structure is called a globin fold

Bohr effect

The regulation of oxygen binding by hydrogen ions and carbon dioxide is called the Bohr effect after Christian Bohr, who described this phenomenon in 1904

Structural Changes at the Heme Groups are Transmitted to the a1b1a2b2 Interface

The transition from deoxyhemoglobin (T state) to oxyhemoglobin (R state) occurs upon oxygen binding. The iron ion moves into the plane of the heme when oxygen binds. The proximal histidine, which is a member of an α-helix, moves with the iron. The resulting structural change is communicated to the other subunits so that the two αβ dimers rotate with respect to one another, resulting in the formation of the R state.

Why are X‑rays the preferred type of electromagnetic radiation used to produce diffraction patterns of protein crystals? X‑rays have the shortest wavelength of all types of electromagnetic radiation. X‑rays do not cause radiation damage to the covalent bonds in biological molecules. Both film and solid state electromagnetic detectors can record X‑ray diffraction patterns. Biological molecules cannot scatter other types of electromagnetic radiation. The wavelength of a typical X‑ray corresponds to the length of a covalent bond.

The wavelength of a typical X‑ray corresponds to the length of a covalent bond.

Shape of curve that communicates that hemoglobin effectively delivers oxygen from the lungs to the tissues

There is a positive relationship between the pO2 and the YO2. Less saturation in tissues than lungs

True or false. A reaction that happens very rarely can be highly spontaneous

True

True or false. Heme is a cofactor necessary to the function of myoglobin

True

True or false. In proteins exhibiting positive cooperativity, small changes in ligand concentration can result in large changes in the proportion of bound sites

True

Total protein determination

UV absorption and colorimetric reactions

Western Blotting

Very small quantities of a protein of interest in a cell or in body fluid can be detected by an immunoassay technique called western blotting. A sample is subjected to electrophoresis on an SDS-polyacrylamide gel. A polymer sheet is pressed against the gel, transferring the resolved proteins on the gel to the sheet, which makes the proteins more accessible for reaction. An antibody that is specific for the protein of interest, called the primary antibody, is added to the sheet and reacts with the antigen. The antibody-antigen complex on the sheet can then be detected by rinsing the sheet with a second antibody, called the secondary antibody, that is specific for the primary antibody (e.g., a goat antibody that recognizes mouse antibodies). Typically, the secondary antibody is fused to an enzyme that produces a chemiluminescent or colored product or contains a fluorescent tag, enabling the identification and quantification of the protein of interest.

Oxygen binding curve

We can determine the oxygen-binding properties of each of these proteins by observing its oxygen-binding curve, a plot of the fractional saturation versus the concentration of oxygen. The fractional saturation, Y, is defined as the fraction of possible binding sites that contain bound oxygen. The value of Y can range from 0 (all sites empty) to 1 (all sites filled). The concentration of oxygen is most conveniently measured by its partial pressure, pO2.For myoglobin, a binding curve indicating a simple chemical equilibrium is observed (Figure 7.7). Notice that the curve rises sharply as pO2 increases and then levels off. Half-saturation of the binding sites, referred to as p50 (for 50% saturated), is at the relatively low value of 2 torr (mm Hg), indicating that oxygen binds with high affinity to myoglobin.

Protein structure determination

X-ray crystallography, NMR spectroscopy, cryogenic electron microscopy (Cryo-EM)

Saturation of O2

YO2^n / YO2^n + P50^n Top: atmosphere/lungs Bottom: tissues

Fluorescence-activated cell sorting (FACS) is a powerful analytical technique that separates cells according to their content of particular molecules. Researchers can use fluorescence-labeled antibodies specific for a cell surface protein to detect cells containing a molecule of interest. Suppose that a researcher wants to isolate cells that possess a receptor that detects and binds bacterial degradation products. However, the researcher does not have an antibody directed against this receptor. Which fluorescence-labeled molecule should the researcher prepare to identify the cells of interest? a derivative of the ligand that binds to the receptor an antibody specific for ligand-gated ion channels an antibody specific for cell surface glycoproteins potassium ions, K+, or sodium ions, Na+

a derivative of the ligand that binds to the receptor

Mass spectrometry

a molecule is ionized into the gas phase and the mass to charge ratio of the resulting ions is determined. Mass spectrometry is a powerful tool to determine the mass and investigate aspects of macromolecular structure

Dimers

a molecule or molecular complex consisting of two identical molecules linked together

Green Fluorescent Protein (GFP)

a naturally fluorescent protein isolated from the jellyfish Aequorea victoria

Homogenate

a suspension of cell fragments and cell constituents obtained when tissue is homogenized; is formed by disrupting the cell membrane, and the mixture is fractionated by centrifugation, yielding a dense pellet of heavy material at the bottom of the centrifuge tube and a lighter supernatant above

Green fluorescent protein (GFP) is a reporter often used to assess gene expression levels or to determine where a protein localizes within a cell. The fusion of GFP to a target protein results in the expression of a chimeric fluorescent protein. Where must the GFP construct be inserted in order for a cell to express a chimeric fluorescent protein? adjacent to the gene of the target protein in the DNA into the target protein's mRNA transcript before the N-terminus of the target protein after the C-terminus of the target protein

adjacent to the gene of the target protein in the DNA

Antigen-specific affinity

affinity purification of only those specific antigens

BPG

allosteric activator prevents R state formation lowers the oxygen affinity of hemoglobin by shifting the T and R equilibrium to the left. Easier O2 delivery to the tissues because there's no O2 in the lungs it'll shift left so that equilibrium can be maintained BPG promotes O2 release - Tense state BPG decreases hemoglobins affinity to O2 and because of that O2 is more easily delivered to the tissues BPG prevents O2 binding, increases O2 delivery

Gel-filtration chromatography

also known as molecular exclusion chromatography (Figure 3.3). The sample is applied to the top of a column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (carbohydrates) or polyacrylamide. Sephadex, Sepharose, and Biogel are commonly used commercial preparations of these beads, which are typically 100 μm (0.1mm) in diameter. Small molecules can enter these beads; large ones cannot. The result is that small molecules are distributed in the aqueous solution both inside the beads and between them, whereas large molecules are located only in the solution between the beads. Large molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them. Molecules of medium size occasionally enter the beads and will flow from the column at an intermediate position, while small molecules, which take a longer, tortuous path, will exit last.

BPG

an allosteric modifier that binds to the central cavity of hemoglobin and affects hemoglobins affinity as well as the shape of the molecule

Western blot

analytical technique. Used to detect, analyze, and quantify proteins - can't recover protein

SDS-PAGE electrophoresis

analytical technique. Used to separate proteins based on their molecular weight. Breaks them apart from their subunits - can't recover protein

How do X‑ray crystallographers reconstruct the crystal structure of a protein following X‑ray diffraction? capturing the undiffracted X‑rays on a detector focusing the X‑rays into an image using lenses applying a Fourier transform to the reflection spots rotating the crystal with respect to the X‑ray source

applying a Fourier transform to the reflection spots

Suppose Gina climbs a high mountain where the oxygen partial pressure in the air decreases to 80 torr. Assume that the pH of her tissues and lungs is 7.4 and the oxygen concentration in her tissues is 20 torr. The P50 of hemoglobin is 26 torr. The degree of cooperativity of hemoglobin, n, is 2.8. Estimate the percentage of the oxygen‑carrying capacity that she utilizes. Calculate your answer to one decimal place. capacity: After Gina spends a day at the mountaintop, where the oxygen partial pressure is 80 torr, the concentration of 2,3‑bisphosphoglycerate (2,3‑BPG) in her red blood cells increases. Why does increasing the concentration of 2,3‑BPG in Gina's blood cells help her function well at high altitudes? Her hemoglobin P50 decreases, causing more blood‑to‑tissue oxygen offloading. The extra 2,3‑BPG stabilizes the relaxed, or R, state of hemoglobin, increasing oxygen binding. Her oxygen‑binding curve shifts to the right, promoting oxygen delivery to tissues. Excess 2,3‑BPG binds oxygen when hemoglobin becomes saturated, acting as an oxygen transporter.

capacity = 63.5 % Her oxygen‑binding curve shifts to the right, promoting oxygen delivery to tissues.

What are the characteristics of a secondary antibody used in Western blotting? contains a covalently attached tag recognizes the Fab region of the primary antibody recognizes antibodies from multiple species recognizes the Fc region of the primary antibody

contains a covalently attached tag, recognizes the Fc region of the primary antibody

A protein has the amino acid sequence DSRLSKTMYSIEAPAKLDWEQNMALDSRLSKTMYSIEAPAKLDWEQNMAL How many peptide fragments would result from cleaving the sequence with each reagent. cyanogen bromide: trypsin: chymotrypsin: Which of these three reagents gives the smallest single fragment (in number of amino acid residues)? trypsin cyanogen bromide chymotrypsin

cyanogen bromide: 3 trypsin: 4 chymotrypsin: 7 chymotrypsin

What factors aid in the release of oxygen from hemoglobin in the peripheral tissues?

decreased concentration of oxygen, increased concentration of carbon dioxide, decreased pH, catalytic activity of carbonic anhydrase, high binding affinity of oxygen to myoglobin, low binding affinity of oxygen to the T-state of hemoglobin, reversible binding of 2,3-BPG to hemoglobin, positive cooperativity of oxygen binding by hemoglobin, ion pairs of hemoglobin's T-state, high levels of catabolic processes in muscle cells

Proteome

derived from proteins expressed by the genome—of an organism signifies a more complex level of information content, encompassing the types, functions, and interactions of proteins within its biological environment.

Chemical shifts

different frequencies shown on a chart

Physicocehmical fractionation

differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on their size, charge, or other shared characteristics of antibodies in a typical sample isolates a subset of sample proteins that include immunoglobulins

Specific protein determination

enzyme activity, binding assays, immunological techniques, bioassays

Applications of gel filtration chromatography

estimation of molecular weights and separation of components in a mixture by size

Samples of the octapeptide AVGWRVKS are subjected to proteolytic cleavage by either trypsin or chymotrypsin. Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by trypsin. ion‑exchange chromatography salting out dialysis gel‑filtration chromatography Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by chymotrypsin. gel‑filtration chromatography salting out dialysis ion‑exchange chromatography

gel-filtration chromatography, ion-exchange chromatography

An IgG antibody that specifically recognizes a protein of interest, p50, was used to isolate p50 from a whole‑cell lysate. The resulting protein mixture can undergo SDS-PAGE to separate p50 from IgG. The molecular weight of p50 is 50 kDa, and the protein does not contain any cysteine residues. The molecular weight of IgG is 160 kDa. IgG contains two heavy chains and two light chains that weigh 50 kDa and 25 kDa, respectively. Both types of chains contain cysteine residues. Select the components of the SDS‑PAGE loading buffer. glycerol sodium chloride sodium dodecyl sulfate dithiothreitol bromophenol blue

glycerol, sodium dodecyl sulfate, bromophenol blue

What functional groups of hemoglobin bind directly to O2

heme and distal histidine

Function of hemoglobin

hemoglobin transports O2 in the blood from the lungs to the tissues. Load O2 in the lungs, unload O2 in the tissues

The average volume of a red blood cell is 87μm3.87μm3. The mean concentration of hemoglobin in red blood cells is 0.34 g⋅ml−1.0.34 g⋅ml−1. What is the weight of the hemoglobin contained in an average red blood cell? hemoglobin weight: How many hemoglobin molecules are there in an average red blood cell? Assume that the molecular weight of the human hemoglobin tetramer is 65 kDa. number of hemoglobin molecules:

hemoglobin weight: 2.96 x 10^-11 g number of hemoglobin molecules: 2.71 x 10^8 molecules

Affinity Chromatography: Identify the buffer solution that can be used for eluting a transcription factor bound to a DNA affinity column. high sugar concentration a mixture of free DNA nucleotides high salt concentration high imidazole concentration

high salt concentration

Hybridoma cells

hybrid cells

Hemoglobin is a protein in red blood cells that binds to oxygen. Which physiological changes that naturally occur in the body reduce hemoglobin's affinity for oxygen? increase in pH increase in temperature accumulation of carbon dioxide accumulation of nitrogen

increase in temperature accumulation of carbon dioxide

What factors aid in the binding of oxygen to hemoglobin in the lungs?

increased concentration of oxygen, decreased concentration of carbon dioxide, increased pH, catalytic activity of carbonic anhydrase, high binding affinity of oxygen to the R-state of hemoglobin, reversible binding of 2,3-BPG to hemoglobin, positive cooperativity of oxygen binding by hemoglobin

Suppose that you are given a mixture of proteins with their properties provided in the following chart. Isoelectric point (pI) Molecular weight (in kDa) Protein A 4.1. 80 Protein B 9.0. 81 Protein C 8.8. 37 Protein D 3.9. 172 Select one combination of techniques that can be used to isolate Protein B from Proteins A, C, and D. gel filtration chromatography and ultracentrifugation dialysis and ion exchange chromatography ion exchange chromatography and gel filtration chromatography dialysis and ultracentrifugation

ion exchange chromatography and gel filtration chromatography

Carbon monoxide (CO)

is a colorless, odorless gas that binds to hemoglobin at the same site as oxygen, forming a complex termed carboxyhemoglobin. Formation of carboxyhemoglobin exerts devastating effects on normal oxygen transport in two ways. First, carbon monoxide binds to hemoglobin about 200-fold more tightly than does oxygen. Even at low partial pressures in the blood, carbon monoxide will displace oxygen from hemoglobin, preventing its delivery. Second, carbon monoxide bound to one site in hemoglobin will shift the oxygen saturation curve of the remaining sites to the left, forcing the tetramer into the R state. This results in an increased affinity for oxygen, preventing its dissociation at tissues.

Affinity chromatography

is another powerful means of purifying proteins that is highly selective for the protein of interest. This technique takes advantage of the high affinity of many proteins for specific chemical groups. For example, the plant protein concanavalin A is a carbohydrate-binding protein, or lectin (Section 11.4), that has affinity for glucose. When a crude extract is passed through a column of beads containing covalently attached glucose residues, concanavalin A binds to the beads, whereas most other proteins do not (Figure 3.5). The bound concanavalin A can then be released from the column by adding a concentrated solution of glucose. The glucose in solution displaces the column-attached glucose residues from binding sites on concanavalin A. Affinity chromatography is a powerful means of isolating transcription factors—proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is passed through a column containing specific DNA sequences attached to a matrix; proteins with a high affinity for the sequence will bind and be retained. In this instance, the transcription factor is released by washing with a solution containing a high concentration of salt.

Gel electrophoresis

is performed in a thin, vertical slab of polyacrylamide gel. Polyacrylamide gels are choice supporting media for electrophoresis because they are chemically inert and readily formed by the polymerization of acrylamide with a small amount of the cross-linking agent methylenebisacrylamide to make a three-dimensional mesh (Figure 3.8). Electrophoresis is distinct from gel filtration in that, because of the electric field, all of the molecules, regardless of size, are forced to move through the same matrix; Small proteins move rapidly through the gel, whereas large proteins stay at the top, near the point of application of the mixture. The mobility of most polypeptide chains under these conditions is linearly proportional to the logarithm of their mass

Nuclear magnetic resonance (NMR) spectroscopy

is unique in being able to reveal the atomic structure of macromolecules in solution, provided that highly concentrated solutions (around 1mM or 15mg ml-) for a 15-kDa protein can be obtained. This technique depends on the fact that certain atomic nuclei are intrinsically magnetic.

The mutated form of hemoglobin (hemoglobin S, or HbS) in sickle‑cell anemia results from the replacement of a glutamate residue by a valine residue at position 66 in the β chain of the protein. Normal hemoglobin is designated HbA. Under conditions of low [O2][O2] , HbS aggregates and distorts the red blood cell into a sickle shape. See image of eight aggregated HbS molecules. Sickled red blood cells are relatively inflexible and may clog capillary beds, causing pain and tissue damage. The sickled red blood cells also have a shorter life span, leading to anemia. Which amino acids would be expected to produce a similar sickling effect if substituted for Val at position 6? phenylalanine arginine lysine leucine alanine Sickling occurs in deoxyhemoglobin S but not in oxyhemoglobin S. Oxyhemoglobin has a small, hydrophobic pocket in a β chain region located in the interior of the protein. In deoxyhemoglobin, however, this pocket is located on the surface of the protein. In deoxyhemoglobin S, Val 66 interacts with this surface pocket, leading to aggregation of HbS. Choose two amino acids that would be reasonable candidates for the pocket-Val 66 interaction. glutamine leucine phenylalanine serine glutamate How does HbS aggregation occur in sickle‑cell anemia? Place the steps in the correct order. Note that deoxyhemoglobin is in the T state; oxyhemoglobin is in the R state. No aggregation [O2] decreases due to vigorous exercise or high altitude.R state Hb shifts to T state Hb. Val interacts with the pocket of a β chain on another HbS. Additional T state HbS interact with the growing aggregate to form an insoluble fiber. Sickled red blood cell

leucine, alanine leucine, phenylalanine No aggregation [O2] decreases due to vigorous exercise or high altitude. R state Hb shifts to T state Hb. Val interacts with the pocket of a β chain on another HbS. Additional T state HbS interact with the growing aggregate to form an insoluble fiber. Sickled red blood cell

The amide hydrogen atoms of peptide bonds within proteins can exchange with protons in a solvent. In general, amide hydrogen atoms in buried regions of proteins and protein complexes exchange more slowly than those on the solvent‑accessible surface. By determining these exchange rates, we can explore protein‑folding reactions, probe the tertiary structure of proteins, and identify the regions of protein-protein interfaces. We can investigate these exchange reactions by studying the behavior of the protein in solvent that has been labeled with deuterium, H2,H2, a stable isotope of hydrogen. Select the methods that could be readily applied to the study of hydrogen-deuterium exchange rates in proteins. mass spectrometry (MS) X-ray crystallography enzyme-linked immunosorbent assay (ELISA) nuclear magnetic resonance (NMR) spectroscopy

mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy)

Which activities often involve the use of reporter constructs? determination of protein function assessment of gene regulation quantification of protein size measurement of gene expression levels identification of a protein's localization

measurement of gene expression levels, identification of a protein's localization

Anion exchange

negatively charged proteins (anionic proteins) can be separated by anion exchange on positively charged diethylaminoethylcellulose (DEAE-cellulose) columns.

Isoelectric Point (pI)

of a protein is the pH at which its net charge is zero (​​electrophoretic mobility is zero)

Homotropic cooperativity

oxygen binding at on site is affecting oxygen binding at another site same ligand, oxygen, is involved at the multiple sites

When YO2 = 0.5, the value of pO2 is defined as?

p50

Draw a new binding curve that is shifted to the right. Label the p50 on the new curve. How does the new value of p50 for your new curve compare to the original p50 value?

pO2 value will shift to the right, YO2 increases. Binding affinity decreases because the concentration goes up while the saturation stays the same

In the lungs, oxygen diffuses into the blood and is loaded onto hemoglobin for transport. In the tissues, oxygen is unloaded from hemoglobin and diffuses from the blood into nearby cells. What drives the diffusion of oxygen? partial pressure of oxygen concentration of ozone body temperature blood pH concentration of carbon dioxide

partial pressure of oxygen

Select all the parameters that can be determined by analyzing a protein sample with tandem mass spectrometry. precursor ion mass isoelectric point amino acid sequence genetic sequence

precursor ion mass, amino acid sequence

Ion-exchange chromatography

proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not (Figure 3.4). The bound protein can then be eluted (released) by increasing the concentration of sodium chloride or another salt in the eluting buffer

Preparative methods

purpose is to isolate and purify a substance from proteins

Polyclonal

referring to the fact that they are derived from multiple antibody-producing cell populations

A protein biochemist attempted to determine the amino acid sequence of a decapeptide. Use the results from the trypsin, chymotrypsin, and cyanogen bromide treatments to suggest the amino acid sequence of this decapeptide. Trypsin digestion gave two fragments with multiple residues (not in order): T1: Ala, Arg, Phe, Gly, Thr, Trp, Tyr T2: Lys, Met, Val Chymotrypsin digestion gave four fragments with multiple residues (not in order): CT1: Ala, Phe CT2: Thr, Trp CT3: Lys, Met, Tyr, Val CT4: Arg, Gly Treatment with cyanogen bromide yielded a single amino acid, methionine, and a nonapeptide. What is a possible sequence of the decapeptide? Use three‑letter abbreviations in your answer, and add a dash between each residue.

sequence: Met-Val-Lys-Tyr-Thr-Trp-Ala-Phe-Gly-Arg

Class-specific affinity

solid-phase binding of particular antibody classes by immobilized ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins purifies all antibodies of the target class without regard to antigen specificity

A nuclear magnetic resonance, or NMR, spectrometer is an instrument that can be used to characterize molecules based upon: the excitation of inner shell electrons. the nuclear radiation emitted by certain molecules. vibrations of portions of the molecule brought about by the absorption of infrared radiation. the behavior of the nuclei of certain atoms in a magnetic field.

the behavior of the nuclei of certain atoms in a magnetic field

All of the cells in the body need oxygen. Hemoglobin molecules in red blood cells transport oxygen through the bloodstream. Oxygen is loaded onto hemoglobin molecules in the lungs and unloaded from the hemoglobin molecules in the tissues. What drives the loading of oxygen onto hemoglobin molecules in the lungs? the high partial pressure of carbon dioxide in the lungs the low partial pressure of oxygen in the lungs the low partial pressure of carbon dioxide in the lungs the high partial pressure of oxygen in the lungs

the high partial pressure of oxygen in the lungs

Structure of myoglobin prevents the release of reactive oxygen species

the interaction between iron and oxygen exists as resonance structures, one with Fe2+ and O2 and another with Fe3+ and O2-. Oxygen can only be released in the O2 state because: - Superoxide is very reactive and can quickly bind to and harm other cellular molecules. - The iron ion would be left in the ferric (Fe3+) state, preventing it from binding additional oxygen. Myoglobin in this state is called metmyoglobin. Features of myoglobin help to prevent superoxide release, including an additional (distal) histidine that donates a hydrogen bond to the bound oxygen molecule.

Report two ways experimental conditions affect how an ion exchange column separation works

the pH of the environment - can cause protonation/deprotonation and the salt concentration of the environment bc the binding will be affected by the salt

What characteristics determine the position of a protein on an IPG strip at the end of isoelectric focusing? the protein's three‑dimensional structure protein solubility the molecular weight of the protein the pI of the protein local pH in the medium

the pI of the protein, local pH in the medium

Nuclear Overhauser enhancement spectroscopy (NOESY) generates two‑dimensional NMR spectra. What unique information can these spectra provide about macromolecules? the proximity of a proton to other protons in the molecule the number of protons in the molecule the spin state energy of the protons in the molecule the number of carbon-hydrogen bonds in the molecule

the proximity of a proton to other protons in the molecule

Specific Activity

the ratio of enzyme activity to the amount of protein in the mixture. Ideally, the specific activity will rise as the purification proceeds and the protein mixture will contain the protein of interest to a greater extent. In essence, the overall goal of the purification is to maximize the specific activity. For a pure enzyme, the specific activity will have a constant value.

Co-immunoprecipitation

the sample of interest is extracted and prepared from cultured cells or isolated tissue, for example—is incubated with the specific antibody. Then, agarose beads coated with an antibody-binding protein (such as Protein A from the bacterium Staphylococcus aureus) are added to the mixture. Protein A recognizes a portion of the antibody that is separate from the antigen-binding region (the Fc domain, Figure 3.17), and thus does not disrupt the protein-antibody complex. After centrifugation at low speed, the antibody, now bound to the beads, aggregates at the bottom of the tube. Under optimal buffer conditions, the antibody-protein complex will also precipitate any additional proteins that are bound to the original protein (Figure 3.24). Subsequent analysis of the precipitate by SDS-PAGE, followed by either western blot or mass spectrometric fingerprinting (Section 3.3), enables the identification of the binding partners.

In H-NMR spectroscopy, which factor determines the shape of the spectra? the energy of a proton's α spin state the number of protons in the macromolecule the transition between a proton's α and β spin states the energy of a proton's β spin state

the transition between a proton's α and β spin states

What are the roles of sodium dodecyl sulfate (SDS) in two‑dimensional electrophoresis? to cause bound proteins to have a large positive charge to denature proteins to cause bound proteins to have a large negative charge to preserve zero net charge of the proteins to preserve the structure of the bound proteins

to denature proteins, to cause bound proteins to have a large negative charge

Assay

to monitor the success of this purification, the biochemist needs a test, called an assay, for some unique identifying property of the protein. A positive result on the assay indicates that the protein is present.

Analytical methods

used to separate the components of a protein

X-ray crystallography

was the first method developed to determine protein structure in atomic detail. This technique provides the clearest visualization of the precise three-dimensional positions of most atoms within a protein. Of all forms of radiation, x-rays provide the best resolution for the determination of molecular structures because their wavelength approximately corresponds to the length of a covalent bond. The three components in an x-ray crystallographic analysis are a protein crystal, a source of x-rays, and a detector

What information do you need about a protein to purify a protein using ion exchange chromatography?

you would need to know the charge, the pI