Biochem Homework #1

Stereoisomers that are nonsuperimposable mirror images of each other are known as: a. anomers. b. cis-trans isomers. c. geometric isomers. d. diastereoisomers. e. enantiomers.

enantiomers.

E. coli is known as a gram-negative bacterial species. (a) How is this determined? (b) How do gram- negative bacteria differ structurally from gram-positive bacteria?

(a) Gram-negative bacteria have little affinity for the dye gentian violet used in Gram's stain, but gram-positive bacteria retain Gram's stain. (b) Gram-negative bacteria have an outer membrane and a peptidoglycan layer; gram-positive bacteria lack an outer membrane and the peptidoglycan layer is much thicker.

For each of these methods of separating proteins, describe the principle of the method, and tell what property of proteins allows their separation by this technique. (a) ion-exchange chromatography(b) size-exclusion (gel filtration) chromatography (c) affinity chromatography

(a) Ion-exchange chromatography separates proteins on the basis of their charges. (b) Size- exclusion or gel filtration chromatography separates on the basis of size. (c) Affinity chromatography separates proteins with specific, high affinity for some ligand (attached to an inert support) from other proteins with no such affinity. (See Fig. 3-17, p. 87.)

You are trying to determine the sequence of a protein that you know is pure. Give the most likely explanation for each of the following experimental observations. You may use a simple diagram for your answer. (a) The Sanger reagent (FDNB, fluorodinitrobenzene) identifies Ala, Gly and Leu as amino-terminal residues, in roughly equal amounts. (b) Your protein splits into two bands with an apparent Mr of 80,000 and 60,000, as determined by SDS- polyacrylamide gel electrophoresis. After treatment of the protein with beta-mercaptoethanol, the same technique reveals three proteins of Mr 35,000, 45,000 and 60,000. (c) Anion exchange chromatography, followed by SDS-PAGE of thefractions collected (under reducing conditions) reveals that the three proteins are within the same SDS-PAGE gel lane. (d) Size-exclusion chromatography (gel filtration) experiments indicate the native protein has an apparent Mr of 420,000.

(a) The protein has some multiple of three subunits, with Ala, Gly and Leu as the amino- terminal residues. (b) One protein has Mr 60,000 and the another one is a disulfide-linked heterodimer that has two subunits (Mr 35,000 and 45,000). (c) The three gel bands that were separated in one lane by SDS-PAGE suggest that the proteins may interact with each other. (d) The native protein (Mr 420,000) is a homotrimer of a heterotrimer (Mr 420,000), that is composed of the three subunits (Mr 35,000, Mr 45,000 and Mr 60,000), two of which are disulfide linked.

You are trying to determine the sequence of a protein that you know is pure. Give the most likely explanation for each of the following experimental observations. You may use a simple diagram for your answer. (a) The Sanger reagent (FDNB, fluorodinitrobenzene) identifies Ala and Leu as amino-terminal residues, in roughly equal amounts. (b) Your protein has an apparent Mr of 80,000, as determined by SDS-polyacrylamide gel electrophoresis. After treatment of the protein with performic acid, the same technique reveals two proteins of Mr 35,000 and 45,000. (c) Size-exclusion chromatography (gel filtration) experiments indicate the native protein has an apparent Mr of 160,000.

(a) The protein has some multiple of two subunits, with Ala and Leu as the amino-terminal residues. (b) The protein has two subunits (Mr 35,000 and 45,000), joined by one or more disulfide bonds. (c) The native protein (Mr 160,000) has two Mr 35,000 subunits and two Mr 45,000 subunits.

Each of the following reagents or conditions will denature a protein. For each, describe in one or two sentences what the reagent/condition does to destroy native protein structure. (a) urea (b) high temperature (c) detergent (d) low pH

(a) Urea acts primarily by disrupting hydrophobic interactions. (b) High temperature provides thermal energy greater than the strength of the weak interactions (hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces, breaking these interactions). (c) Detergents bind to hydrophobic regions of the protein, preventing hydrophobic interactions among several hydrophobic patches on the native protein. (d) Low pH causes protonation of the side chains of Asp, Glu, and His, preventing electrostatic interactions.

In one or two sentences, describe the usefulness of each of the following reagents or reactions in the analysis of protein structure: (a) Edman reagent (phenylisothiocyanate) (b) Sanger reagent (1-fluoro-2,4-dinitrobenzene, FDNB) (c) trypsin

(a) used in determination of the amino acid sequence of a peptide, starting at its amino terminus; (b) used in determination of amino-terminal amino acid of a polypeptide; (c) used to produce specific peptide fragments from a polypeptide.

At pH 3, aspartic acid (pKs are alpha-carboxylate 1.99, alpha-amino 9.9, beta-carboxylate 3.9 ) would be charged as follows: a. -1 alpha-carboxylate, +1 alpha-amino, -1 beta-carboxylate, -1 net charge b. -1 alpha-carboxylate, +1 alpha-amino, 0 beta-carboxylate, 0 net charge c. +1 alpha-carboxylate, +1 alpha-amino, +1 beta-carboxylate, +3 net charge d. alpha-carboxylate, -1 alpha-amino, 0 beta-carboxylate, -1 net charge e. alpha-carboxylate, +1 alpha-amino, +1 beta-carboxylate, +2 net charge

-1 alpha-carboxylate, +1 alpha-amino, 0 beta-carboxylate, 0 net charge

The pH of a solution of 1 M HCl is: a. 1 b. 10 c. 0 d. -1 e. 0.1

0

A polypeptide is hydrolyzed, and it is determined that there are 3 Lys residues and 2 Arg residues (as well as other residues). How many peptide fragments can be expected when the native polypeptide is incubated with the proteolytic enzyme trypsin?

6

The pH of a sample of blood is 7.4, while gastric juice is pH 2.4. The blood sample has: a. 100,000 times lower [H+] than the gastric juice. b. 0.189 times the [H+] as the gastric juice. c. 6 times lower [H+] than the gastric juice. d. 5.29 times lower [H+] than the gastric juice. e. a million times lower [H+] than the gastric juice.

100,000 times lower [H+] than the gastric juice.

Roughly how many amino acids are there in one turn of an ??helix? a. 4.2 b. 1.9 c. 3.8 d. 3.6 e. 10

3.6

Define a chiral center

A chiral center is a carbon atom that has four different substituents attached, and cannot be superimposed on its mirror image - as a right hand cannot fit into a left glove.

Titrate Ala in non-ionic form at neutral pH. Show the graph.

Answer: Not possible

hydronium ion: a. is the usual form of one of the dissociation products of water in solution. b. is a hydrated proton. c. All of the above are true. d. is a hydrated hydrogen ion. e. has the structure H3O+.

All of the above are true.

Describe a purification procedure for a mitochondrial (membrane or soluble) protein from 1) spinach leaves 2) dog brain and 3) cat fur. USE ALL BIOCHEMICAL METHODS THAT ARE DESCRIBED IN CHAPTERS 1-4 AND IN THE ADDITIONAL MATERIAL PROVIDED. If you need 2 or even 10 pages, write on additional pages.

Ans: Take the leaves, put in a blender, and start with sufficient volume to have enough final product. The leaves should be subjected to gentle blending or grinding with neutral buffer, all the steps will be done on ice. Subject it to low speed centrifugation, discard pellet and collect supernatant. 2nd centrifugation at a higher speed, followed by 3rd very strong centrifugation, where all the chloroplast goes to the pellet. Add percoll or sucrose gradient which will separate the cells and chloroplasts in a gradient. Based on the gradient, cells will be at the top, intact mitochondria in the middle and intact chloroplasts at the bottom. Use a suction device and get each layer out into their respective vials. Dialyze them in a dialysis bag. Take the mitochondria fraction, break it by osmotic shock. Centrifuge and the membrane goes to the bottom and soluble part will be in the supernatant. To purify the soluble protein, do an ion exchange chromatography and gel filtration. Then to verify - do electrophoresis with the controls from every step. Finally do MS analysis for sequence information. (B) Dissect brains and, place on ice & cut into small pieces and grind it. Gently homogenize the tissue. 20 Centrifuge for few minutes to separate the cytoplasm (supernatant). Rest of the steps are same as above. (C) Cat fur: Not possible.

A biochemist wishes to determine the sequence of a protein that contains 123 amino acid residues. After breaking all of the disulfide bonds, the protein is treated with cyanogen bromide (CNBr), and it is determined that that this treatment breaks up the protein into seven conveniently sized peptides, which are separated from each other. It is your turn to take over. Outline the steps you would take to determine, unambiguously, the sequence of amino acid residues in the original protein.

Ans: (1) Use Edman degradation to determine the sequence of each peptide (2) Create a second set of peptides by treatment of the protein with a specific protease (for example, trypsin), and determine the sequence of each of these. (3) Place the peptides in order by their overlaps. (4) Finally, by a similar analysis of the original protein without first breaking disulfide bonds, determine the number and location of —S—S— bridges An alternate answer is determining the sequence by Mass Spectrometry

Briefly define "isotonic," "hypotonic," and "hypertonic" solutions. (b) Describe what happens when a cell is placed in each of these types of solutions.

Ans: (a) An isotonic solution has the same osmolarity as the solution to which it is being compared. A hypotonic solution has a lower osmolarity than the solution to which it is being compared. A hypertonic solution has a higher osmolarity than the solution to which it is being compared. (b) Higher osmolarity results in osmotic pressure, which generally leads to movement of water across a membrane. In an isotonic solution, in which the osmolarity of the solution is the same as the cell cytoplasm, there will be no net water movement. In a hypotonic solution, water will move into the cell, causing the cell to swell and possibly burst. In a hypertonic solution, water will move out of the cell and it will shrink.

A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties: (a) What type of protein separation techniques might she use to separate (b) protein X from protein A? (c) protein X from protein B? (d) protein X from protein C?

Ans: (a) size-exclusion (gel filtration) chromatography to separate on the basis of size; (b) ion- exchange chromatography or isoelectric focusing to separate on the basis of charge; (c) specific affinity chromatography, using immobilized DNA.

Any given protein is characterized by a unique amino acid sequence (primary structure) and three- dimensional (tertiary) structure. How are these related?

Ans: Anfinsen showed that a completely denatured enzyme (ribonuclease) could fold spontaneously into its native, enzymatically active form with only the primary sequence to guide it.

Once a protein has been denatured, how can it be renatured? If renaturation does not occur, what might be the explanation?

Ans: Because a protein may be denatured through the disruption of hydrogen bonds and hydrophobic interactions by salts or organic solvents, removal of those conditions will reestablish the original aqueous environment, often permitting the protein to fold once again into its native conformation. If the protein does not renature, it may be because the denaturing treatment removed a required prosthetic group, or because the normal folding pathway requires the presence of a polypeptide chain binding protein or molecular chaperone. The normal folding pathway could also be mediated by a larger polypeptide, which is then cleaved (for example, insulin). Denatured insulin would not refold easily.

Describe the principles, similarities and differences between Blue Native PAGE and Colorless Native PAGE

Ans: Blue Native-PAGE - Separation is according to the molecular mass of the native protein complexes, due to the external charge induced by Coomassie dye.Colorless Native-PAGE - Separation is according to the internal charge of the native protein complexes, there is no external charge induced by Coomassie dye and no separation according to the molecular mass

How can changes in pH alter the conformation of a protein? Given a random protein, describe the conformations that this protein may have in acidic vs alkaline pH. Use a 15 kDa protein as an example and investigate its conformations in ESI-MS spectra acquired in positive and negative ionization modes (show these spectra).

Ans: Changes in pH can influence the extent to which certain amino acid side chains (or the amino and carboxyl termini) are protonated. The result is a change in net charge on the protein, which can lead to electrostatic attractions or repulsions between different regions of the protein. The final effect is a change in the protein's three-dimensional shape or even complete denaturation. The protein will have more conformations at an acidic pH mainly due to 3 amino acids (K,R,H) available for protonation compared to alkaline Ph. More protonation leads to more charges and hence more conformations. Where as in alkaline pH - there are only two amino acids (D,E) that are available for deprotonation. Look at chapter 3-03; slides 23 and 26 - myoglobin in positive and negative mode. Positive mode - has more charges/ more peaks and negative mode - less charges/ less peaks.

A biochemist is attempting to isolate a cytosolic heterodimeric enzyme from MCF breast cancer cells grown in cell culture. The heterodimer is held together through hydrophobic interactions and is composed of a 129 kDa protein A (pI 4.5) and a 12 kDa protein B (pI 6.9). Design a purification scheme for this heterodimeric protein and justify each step that you will use. USE ALL BIOCHEMICAL METHODS THAT ARE DESCRIBED IN CHAPTERS 1-4 AND IN THE ADDITIONAL MATERIAL PROVIDED. If you need 2 or even 10 pages, write on additional pages.

Ans: Collection of the cells, cell lysis and extraction of proteins. Chromatography (hydrophobic interactions chromatography) to separate subunits of proteins. Separation of subunits using 2D electrophoresis (first according to isolectric point, then according to molecular weight). Alternatively, refer to Dr. Darie's video for this question. The explanation is given in detail on moodle.

Explain why living organisms are able to produce particular chiral forms of different biomolecules while laboratory chemical synthesis usually produces a racemic mixture. Is using a racemic good? If one wants to use only one enantiomer, how would he/she do it?

Ans: Laboratory syntheses usually use achiral reagents and thus produce racemic mixtures of products. In contrast, because all enzymes are made of chiral precursors, all enzymes are inherently chiral catalysts. Thus, they will show strong stereoselectivity in reactants and mechanisms, leading to the production of chiral products. Using a racemic is not good as biomolecules such as receptors for drugs are stereospecific, so each of the two enantiomers of the drug may have very different effects on an organism. One may be beneficial, the other toxic; or one enantiomer may be ineffective and its presence could reduce the efficacy of the other enantiomer. He/She would have to separate the two enantiomers by using optically active reagent and test each for its biological activity and choose the one that is less toxic and more beneficial.

List at least four physico-chemical properties of proteins used in protein purification. Now list five guidelines that are used for protein purification

Ans: Physico-chemical properties of proteins: charge, polarity, hydrophobicity, solubility, isoelectric point, molecular mass, affinity, enantioselectivity, post-translational modificationsGuidelines that are used for protein purification: define objectives, define properties of the target protein and critical impurities, develop analytical assays, minimize sample handling at every stage, minimize use of additives, remove damaging contaminants early, use different technique at each step, minimize number of steps.

Give the general Henderson-Hasselbalch equation and sketch the plot it describes (pH against amount of NaOH added to a weak acid). On your curve label the pKa for the weak acid, and indicate the region in which the buffering capacity of the system is greatest.

Ans: The inflection point, which occurs when the weak acid has been exactly one-half titrated with NaOH, occurs at a pH equal to the pKa of the weak acid. The region of greatest buffering capacity (where the titration curve is flattest) occurs at pH values of pKa ±1. (See Fig. 2-17, p. 59.)

How does one determine the three-dimensional structure of a protein? Describe the principle of the method.

Ans: The protein is crystallized, and the crystal structure is determined by x-ray diffraction. The pattern of diffracted x-rays yields, by Fourier transformation, the three-dimensional distribution of electron density. By matching electron density with the known sequence of amino acids in the protein, each region of electron density is identified as a single atom. Sometimes, the three- dimensional structure of a small protein or peptide can be determined in solution by sophisticated analysis of the NMR spectrum of the polypeptide. This technique can also reveal dynamic aspects of protein structure such as conformational changes. Computer analysis of two-dimensional NMR spectra can be used to generate a picture of the three-dimensional structure of a protein.

The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all; more than one reagent may be suitable for a given purpose.(a) CNBr (cyanogen bromide) (b) Edman reagent (phenylisothiocyanate)(c) FDNB(d) dithiothreitol___ hydrolysis of peptide bonds on the carboxyl side of Lys and Arg ___ cleavage of peptide bonds on the carboxyl side of Met ___ breakage of disulfide (—S—S—) bonds (e) performic acid (f) chymotrypsin (g) trypsin ___ determination of the amino acid sequence of a peptide___ determining the amino-terminal amino acid in a polypeptide

Ans: g; a; d and e; b; c

Draw a titration curve for a peptide with the amino acid sequence Lys-Ser-Glu and now show the ionic form that predominates at pH 4.0 and pH 8.0.

At pH 4 - The carboxylic side chain of glutamate is protonated because the pKa is 4.25 and the pKa of the 2nd carboxylic group is 2.19, hence it will be deprotonated.

Phosphoric acid is tribasic, with pKa's of 2.14, 6.86, and 12.4. The ionic form that predominates at pH 3.2 is: a. H2PO4-. b. HPO42-. c. PO43-. d. H3PO4. e. None of the above

H2PO4-.

Compare the following sequences taken from four different proteins, and select the answer that best characterizes their relationships. A B C1 DVEKGKKIDIMKCS HTVEKGGKHKTGPNLH GLFGRKTGQAPGYSYT2 DVQRALKIDNNLGQ HTVEKGAKHKTAPNVH GLADRIAYQAKATNEE3 LVTRPLYIFPNEGQ HTLEKAAKHKTGPNLH ALKSSKDLMFTVINDD4 FFMNEDALVARSSN HQFAASSIHKNAPQFH NLKDSKTYLKPVISE a. Based only on sequences in column B, protein 4 reveals the greatest evolutionary divergence. b. Protein 4 is the protein that shows the greatest overall homology to protein 1. c. Comparing proteins 1 and 2 in column A reveals that these two proteins have diverged the most throughout evolution. d. The portions of amino acid sequence shown suggest that these proteins are completely unrelated. e. Proteins 2 and 3 show a greater evolutionary distance than proteins 1 and 4.

Based only on sequences in column B, protein 4 reveals the greatest evolutionary divergence.

Which of the following statements is false? a. Silk fibroin is a protein in which the polypeptide is almost entirely in the beta conformation. b. Gly residues are particularly abundant in collagen. c. Collagen is a protein in which the polypeptides are mainly in the alpha-helix conformation. d. Alpha-keratin is a protein in which the polypeptides are mainly in the alpha-helix conformation. e. Disulfide linkages are important for keratin structure.

Collagen is a protein in which the polypeptides are mainly in the alpha-helix conformation.

Differentiate between configuration and conformation.

Configuration denotes the spatial arrangement of the atoms of a molecule that is conferred by the presence of either double bonds, around which there is no freedom of rotation, or chiral centers, which give rise to stereoisomers. Configurational isomers can only be interconverted by temporarily breaking covalent bonds. Conformation refers to the spatial arrangement of substituent groups that, without breaking any bonds, are free to assume different positions in space because of the freedom of bond rotation.

Which of the following statements about cystine is correct? a. Cystine is an example of a nonstandard amino acid, derived by linking two standard amino acids. b. Cystine forms when the —CH2—SH R group is oxidized to form a —CH2—S—S—CH2— disulfide bridge between two cysteines. c. Two cystines are released when a —CH2—S—S—CH2— disulfide bridge is reduced to —CH2—SH. d. Cystine is formed through a peptide linkage between two cysteines. e. Cystine is formed by the oxidation of the carboxylic acid group on cysteine.

Cystine forms when the —CH2—SH R group is oxidized to form a —CH2—S—S—CH2— disulfide bridge between two cysteines.

Describe at least three types of centrifugation, chromatography & electrophoresis methods that are used in protein isolation & purification. Define the principle of each example in 1-2 lanes/method.

Differential centrifugation, step gradient centrifugation, linear gradient centrifugation Ion exchange, gel filtration, affinity, hydrophobic interactions, reverse phase chromatography SD-PAGE, 2D-PAGE, BN-PAGE, CN-PAGE, etc Principle: Refer the slides

Give examples of 5 types of isomers.

Enantiomer, diastereogmer, L Isomer/D Isomer, L Isoleucine, D-Isoleucine, Cis-Isomer, Trans-Isomer

A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly) 2, (Phe) 2, His, Leu, Met. The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC. When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered. Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys. 2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide. Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide. The tetrapeptide was composed of (Lys) 2, Phe, and Gly. The native sequence was determined to be: a. His-Leu-Phe-Gly-Lys-Lys-Phe-Met-Gly. b. Gly-Phe-Lys-Lys-Gly-Leu-Met-Phe-His. c. Met-Leu-Phe-Lys-Phe-Gly-Gly-Lys-His. d. His-Phe-Leu-Gly-Lys-Lys-Phe-Met-Gly. e. His-Leu-Gly-Lys-Lys-Phe-Phe-Gly-Met.

His-Leu-Phe-Gly-Lys-Lys-Phe-Met-Gly.

Describe the principle of hydrophobic interactions chromatography; now describe the principle of hydrophilic interactions chromatography.

Hydrophobic Interaction Chromatography: Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer In hydrophilic interaction chromatography, is a liquid chromatography technique that uses a polar stationary phase in conjunction with a mobile phase containing an appreciable quantity of water combined with a higher proportion of a less polar solvent. Here, the hydrophilic, polar, and charged compounds are retained preferentially compared with hydrophobic neutral compounds

How does the shape of a titration curve confirm the fact that the pH region of greatest buffering power for an amino acid solution is around its pK's?

In a certain range around the pKa's of an amino acid, the titration curve levels off. This indicates that for a solution with pH pK, any given addition of base or acid equivalents will result in the smallest change in pH—which is the definition of a buffer.

Describe the principle of reversed phase chromatography; now describe the principle of normal phase chromatography.

In reversed phase chromatography, the most polar compounds elute first with the most nonpolar compounds eluting last. The mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol, acetonitrile. Retention increases as the amount of the polar solvent (water) in the mobile phase increases. In normal-phase chromatography, the least polar compounds elute first and the most polar compounds elute last. The mobile phase consists of a nonpolar solvent such as hexane or heptane mixed with a slightly more polar solvent such as isopropanol, ethyl acetate or chloroform. Retention decreases as the amount of polar solvent in the mobile phase increases.

Which of the following statements concerning the process of spontaneous folding of proteins is false? a. It may involve a gradually decreasing range of conformational species. b. It may involve initial formation of a highly compact state. c. It may be defective in some human diseases. d. It may be an essentially random process. e. It may involve initial formation of local secondary structure.

It may be an essentially random process.

At pH 7.0, converting a glutamic acid to gamma-carboxyglutamate will have what effect on the overall charge of the protein containing it? a. There is not enough information to answer the question b. It will become more negative c. It will become more positive. d. The answer depends on the salt concentration. e. It will stay the same

It will become more negative, It will stay the same

At pH 7.0, converting a proline to hydroxyproline will have what effect on the overall charge of the protein containing it? a. It will become more negative b. There is not enough information to answer the question. c. It will stay the same. d. the answer depends on the salt concentration. e. It will become more positive.

It will stay the same.

Major advance in the application of mass spectrometry to macromolecules came with the development of techniques to overcome which of the following problems? a. Most macromolecules could not be purified to the degree required for mass spectrometric analysis. b. Mass spectrometric analysis involved molecules in the gas phase. c. Macromolecules were insoluble in the solvents used in mass spectrometry. d. Mass spectrometric analyses of macromolecules were too complex to interpret. e. The specialized instruments required were prohibitively expensive.

Mass spectrometric analysis involved molecules in the gas phase.

Which of the following statements about aromatic amino acids is correct? a. Histidine's ring structure results in its being categorized as aromatic or basic, depending on pH. b. The presence of a ring structure in its R group determines whether or not an amino acid is aromatic. c. The major contribution to the characteristic absorption of light at 280 nm by proteins is the phenylalanine R group. d. All are strongly hydrophilic. e. On a molar basis, tryptophan absorbs more ultraviolet light than tyrosine.

On a molar basis, tryptophan absorbs more ultraviolet light than tyrosine.

Which of the following is not known to be involved in the process of assisted folding of proteins? a. Heat shock proteins b. Peptide bond isomerization c. Chaperonins d. Disulfide interchange e. Peptide bond hydrolysis

Peptide bond hydrolysis

All of the following are considered "weak" interactions in proteins, except a. Van der Waals forces b. Peptide bonds c. Hydrophobic interactions d. Ionic bonds e. Hydrogen bonds

Peptide bonds

Which of the following is correct with respect to the amino acid composition of proteins? a. Larger proteins have a more uniform distribution of amino acids than smaller proteins. b. The average molecular weight of an amino acid in a protein increases with the size of the protein. c. Proteins with the same molecular weight have the same amino acid composition. d. Proteins contain at least one each of the 20 different standard amino acids. e. Proteins with different functions usually differ significantly in their amino acid composition.

Proteins with different functions usually differ significantly in their amino acid composition.

List three examples (of each category; total of 9) of drugs that are sold as 1) racemics, 2) S-enantiomers and 3) R-enantiomers. Now give two examples of optical enantiomers. DO NOT USE EXAMPLES FROM THE BOOK OR THE MATERIAL THAT WAS PRESENTED DURING THE CLASS OR IN THE SUPPLEMENTAL MATERIAL.

Racemic drugs Amlodipine Modafinil Omeprazole S- enantiomer drugs Dexchlorpheniramine Esomeprazole Eszopiclone R- enantiomer drugs Thalidomide Albuterol Methadone Optical enantiomers Isoflurane Hyoscine

One method used to prevent disulfide bond interference with protein sequencing procedures is: a. removing cystines from protein sequences by proteolytic cleavage. b. Reducing disulfide bridges and preventing their re-formation by further modifying the —SH groups. c. sequencing proteins that do not contain cysteinyl residues. d. protecting the disulfide bridge against spontaneous reduction to cysteinyl sulfhydryl groups. e. cleaving proteins with proteases that specifically recognize disulfide bonds.

Reducing disulfide bridges and preventing their re-formation by further modifying the —SH groups.

Why do smaller molecules elute after large molecules when a mixture of proteins is passed through a size-exclusion (gel filtration) column?

The column matrix is composed of cross-linked polymers with pores of selected sizes. Smaller molecules can enter pores in the polymer beads from which larger molecules would be excluded. Smaller molecules therefore have a larger three-dimensional space in which to diffuse, making their path through the column longer. Larger molecules migrate faster because they pass directly through the column, unhindered by the bead pores.

The average molecular weight of the 20 standard amino acids is 138, but biochemists use 110 when estimating the number of amino acids in a protein of known molecular weight. Why? a. The number 138 represents the molecular weight of conjugated amino acids. b. The number 110 reflects the number of amino acids found in the typical small protein, and only small proteins have their molecular weight estimated this way. c. The number 110 reflects the higher proportion of small amino acids in proteins, as well as the loss of water when the peptide bond forms. d. The number 110 is based on the fact that the average molecular weight of a protein is 110,000 with an average of 1,000 amino acids. e. The number 110 takes into account the relatively small size of nonstandard amino acids.

The number 110 reflects the higher proportion of small amino acids in proteins, as well as the loss of water when the peptide bond forms.

Describe the Lexapro vs Celexa study (hypothesis, design, experimental, results and conclusions)

They are both prescription drugs used for depression and both belong to a class of medications called selective serotonin reuptake inhibitors (SSRIs). As SSRIs, their mood-elevating effects are primarily the result of increased levels of serotonin in the brain. They differ in chemical structure, uses, effectiveness and some side effects. Celexa is a mixture of 2 similar, but not identical, types of citalopram molecules called enantiomers. The enantiomers in Celexa are called R-citalopram and S-citalopram.Lexapro was developed from Celexa. It contains just the S-citalopram enantiomer, which is the reason for its generic name _es_citalopram. Lexapro was developed because researchers discovered that S-citalopram was a more effective antidepressant than R-citalopram. Both Lexapro and Celexa are approved by the U.S. Food and Drug Administration (FDA) for the treatment of major depressive disorder 34. Lexapro is also approved for generalized anxiety disorder, whereas Celexa is only approved for depression. This doesn't necessarily mean that Celexa cannot help with anxiety -- it simply means that Celexa has not undergone formal assessment by the FDA to ensure that its benefits outweigh its risks when used to treat anxiety. Even though the usual dose of Lexapro is about one-half of the usual dose for Celexa, Lexapro is generally more effective than Celexa for treating major depression. A systematic review of several previous studies published by the Norwegian Institute of Public Health in 2007 reported that depression was more likely to improve with Lexapro than with Celexa. A more recent systematic review published in the "Cochrane Database of Systematic Reviews" in July 2012 also compared Lexapro and Celexa for the treatment of major depressive disorder. The results showed that improvement was about 1.5 times more likely with Lexapro than with Celexa.

Which of the following statements concerning protein domains is true? a. They have been found only in prokaryotic proteins. b. They may retain their correct shape even when separated from the rest of the protein. c. They are a form of secondary structure. d. They consist of separate polypeptide chains (subunits). e. They are examples of structural motifs.

They may retain their correct shape even when separated from the rest of the protein.

Which of the following statements about buffers is true? a. The strongest buffers are those composed of strong acids and strong bases. b. When pH = pKa, the weak acid and salt concentrations in a buffer are equal. c. A buffer composed of a weak acid of pKa = 5 is stronger at pH 4 than at pH 6. d. The pH of a buffered solution remains constant no matter how much acid or base is added to the solution. e. At pH values lower than the pKa, the salt concentration is higher than that of the acid.

When pH = pKa, the weak acid and salt concentrations in a buffer are equal.

What factors would make it difficult to interpret the results of a gel electrophoresis of proteins in the absence of sodium dodecyl sulfate (SDS)?

Without SDS, protein migration through a gel would be influenced by the protein's intrinsic net charge—which could be positive or negative—and its unique three-dimensional shape, in addition to its molecular weight. Thus, it would be difficult to ascertain the difference between proteins based upon a comparison of their mobilities in gel electrophoresis.

The positive charge on proteins in electrospray ionization mass spectrometry is the result of: a. protonated Arg and Lys residues. b. electrons fired at the gas-phase protein molecules. c. a low pH. d. protons fired at the gas-phase protein molecules. e. protonated Asp and Glu residues.

a low pH.

For amino acids with neutral R groups, at any pH below the pI of the amino acid, the population of amino acids in solution will have: a. no net charge. b. no charged groups. c. positive and negative charges in equal concentration. d. a net negative charge. e. a net positive charge.

a net positive charge.

The major carrier of chemical energy in all cells is: a. adenosine triphosphate. b. cytosine tetraphosphate. c. adenosine monophosphate. d. acetyl triphosphate. e. uridine diphosphate. f. FedEx

adenosine triphosphate.

In an helix, the R groups on the amino acid residues: a. generate the hydrogen bonds that form the helix. b. are found on the outside of the helix spiral. c. cause only right-handed helices to form. d. stack within the interior of the helix. e. alternate between the outside and the inside of the helix.

are found on the outside of the helix spiral.

In the helix the hydrogen bonds: a. occur only near the amino and carboxyl termini of the helix. b. are roughly parallel to the axis of the helix. c. occur only between some of the amino acids of the helix. d. occur mainly between electronegative atoms of the R groups. e. are roughly perpendicular to the axis of the helix.

are roughly parallel to the axis of the helix.

Humans usually wish each other happy birthday, happy holiday, or nice weekend, but some of them wish to their colleagues to have the lowest entropy. This wish usually means that they want their colleagues to: a. have a great activation energy b. have a dynamic steady state. c. be organized d. have a lower metabolism e. be disorganized f. have more free energy

be organized

The bacterium E. coli requires simple organic molecules for growth and energy—it is therefore a: a. photoautotroph. b. chemoheterotroph. c. chemoautotroph. d. phototraph. e. lithograph. f. lithotroph.

chemoheterotroph.

By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to: a. preserve a protein's native structure and biological activity. b. determine an enzyme's specific activity. c. determine the amino acid composition of the protein. d. separate proteins exclusively on the basis of molecular weight. e. determine a protein's isoelectric point.

determine a protein's isoelectric point., separate proteins exclusively on the basis of molecular weight.

The most important contribution to the stability of a protein's conformation appears to be the: a. sum of free energies of formation of many weak interactions between its polar amino acids and surrounding water. b. entropy increase from the increase in ordered water molecules forming a solvent shell around it. c. entropy increase from the decrease in ordered water molecules forming a solvent shell around it. d. stabilizing effect of hydrogen bonding between the carbonyl group of one peptide bond and the amino group of another. e. maximum entropy increase from ionic interactions between the ionized amino acids in a protein.

entropy increase from the decrease in ordered water molecules forming a solvent shell around it.

If the free energy change ?G for a reaction is -46.11 kJ/mol, the reaction is: a. exothermic. b. exergonic. c. at equilibrium. d. endergonic. e. endothermic.

exergonic.

Of the 20 standard amino acids, only ___________ is not optically active. The reason is that its side chain ___________. a. glycine; is unbranched b. alanine; is a simple methyl group c. glycine; is a hydrogen atom d. lysine; contains only nitrogen e. proline; forms a covalent bond with the amino group

glycine; is a hydrogen atom

The three-dimensional structure of macromolecules is formed and maintained primarily through noncovalent interactions. Which one of the following is not considered a noncovalent interaction? a. hydrogen-carbon bonds b. hydrogen bonds c. hydrophobic interactions d. ionic interactions e. van der Waals interactions

hydrogen-carbon bonds

An average protein will be denatured by the compounds below, except: a. urea. b. heating to 90°C. c. a detergent such as sodium dodecyl sulfate. d. iodoacetic acid e. pH 10

iodoacetic acid

The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step: a. proteins with similar isoelectric points become further separated according to their molecular weights. b. the proteins in the bands separate more completely because the second electric current is in the opposite polarity to the first current. c. the individual bands undergo a second, more intense isoelectric focusing. d. proteins with similar isoelectric points can become free of charge e. the individual bands become stained so that the isoelectric focus pattern can be visualized.

proteins with similar isoelectric points become further separated according to their molecular weights.

Consider an acetate buffer, initially at the same pH as its pKa (4.76). When sodium hydroxide (NaOH) is mixed with this buffer, the: a. sodium acetate formed precipitates because it is less soluble than acetic acid. b. pH remains constant. c. pH rises more than if an equal amount of NaOH is added to unbuffered water at pH 4.76. d. ratio of acetic acid to sodium acetate in the buffer falls. e. pH rises more than if an equal amount of NaOH is added to an acetate buffer initially at pH 6.76.

ratio of acetic acid to sodium acetate in the buffer falls.

The Henderson-Hasselbalch equation: a. allows the graphic determination of the molecular weight of a weak acid from its pH alone. b. does not explain the behavior of di- or tri-basic weak acids c. relates the pH of a solution to the pKa and the concentrations of acid and conjugate base. d. employs the same value for pKa for all weak acids. e. is equally useful with solutions of acetic acid and of hydrochloric acid.

relates the pH of a solution to the pKa and the concentrations of acid and conjugate base.

In a mixture of the five proteins listed below, which should elute second in size-exclusion (gel- filtration) chromatography under reducing conditions? a. ribonuclease A Mr = 13,700 b. RNA polymerase Mr = 450,000 c. immunoglobulin G Mr = 145,000 d. serum albumin Mr = 68,500 e. cytochrome c Mr = 13,000

serum albumin Mr = 68,500

The enzyme fumarase catalyzes the reversible hydration of fumaric acid to l-malate, but it will not catalyze the hydration of maleic acid, the cis isomer of fumaric acid. This is an example of: a. stereoisomerization. b. stereospecificity. c. biological activity. d. racemization. e. chiral activity.

stereospecificity.

Experiments on denaturation and renaturation after the reduction and reoxidation of the —S—S— bonds in the enzyme ribonuclease (RNase) have shown that: a. the completely unfolded enzyme, with all —S—S— bonds broken, is still enzymatically active. b. folding of denatured RNase into the native, active conformation requires the input of energy in the form of heat. c. the primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure. d. native ribonuclease does not have a unique secondary and tertiary structure. e. the enzyme, dissolved in water, is thermodynamically stable relative to the mixture of amino acids whose residues are contained in RNase.

the primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure.

Titration of valine by a strong base, for example NaOH, reveals two pK's. The titration reaction occurring at pK2 (pK2 = 9.62) is: a. —COOH + —NH2 —COO + —NH2+. b. —COOH + OH —COO + H2O. c. —NH2 + OH —NH + H2O. d. —COO + —NH2+ —COOH + —NH2. e. —NH3+ + OH —NH2 + H2O.

—NH3+ + OH —NH2 + H2O.