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Lehninger Principles of Biochemistry

David L. Nelson, Michael M. Cox

Chapter 3

Amino Acids, Peptides, and Proteins - all with Video Answers

Educators

Chapter Questions

02:33

Problem 1

The citrulline isolated from watermelons has the structure shown below. Is it $a$D- or L-amino acid? Explain.

Prashant Bana
Prashant Bana
Numerade Educator
07:18

Problem 2

A $100 \mathrm{mL}$ solution of $0.1 \mathrm{M}$ glycine at $\mathrm{pH} 1.72$ was titrated with $2 \mathrm{M} \mathrm{NaOH}$ solution. The $\mathrm{pH}$ was monitored and the results were plotted as shown in the graph. The key points in the titration are designated I to
V. For each of the statements (a) to (o), identify the appropriate key point in the titration and justify your choice.
(a) Glycine is present predominantly as the species $+\mathrm{H}_{3} \mathrm{N}-\mathrm{CH}_{2}-\mathrm{COOH}$
(b) The average net charge of glycine is $+\frac{1}{2}$
(c) Half of the amino groups are ionized.
(d) The pH is equal to the $\mathrm{p} K_{\mathrm{a}}$ of the carboxyl group.
(e) The $\mathrm{pH}$ is equal to the $\mathrm{p} K_{\mathrm{a}}$ of the protonated amino
group.
(f) Glycine has its maximum buffering capacity.
(8) The average net charge of glycine is zero.
(h) The carboxyl group has been completely titrated (first equivalence point
(i) Giycine is completely titrated (second equivalence
point)
(j) The predominant species is $^{+} \mathrm{H}_{3} \mathrm{N}-\mathrm{CH}_{2}-\mathrm{COO}^{-}$
(k) The average net charge of glycine is -1
(1) Giycine is present predominantly as a 50: 50 mixture of $^{+} \mathrm{H}_{3} \mathrm{N}-\mathrm{CH}_{2}-\mathrm{COOH}$ and $^{+} \mathrm{H}_{3} \mathrm{N}-\mathrm{CH}_{2}-\mathrm{COO}^{-}$
(m) This is the isoelectric point.
(n) This is the end of the titration.
(o) These are the worst pH regions for buffering power.

AB
Amanda Bates
Numerade Educator
03:15

Problem 3

At a pH equal to the isoelectric point of alanine, the net charge on alanine is zero. Two structures can be drawn that have a net charge of zero, but the predominant form of alanine at its pI is zwitterionic.
(a) Why is alanine predominantly zwitterionic rather than completely uncharged at its pI?
(b) What fraction of alanine is in the completely uncharged form at its pI? Justify your assumptions.

David Collins
David Collins
Numerade Educator
08:58

Problem 4

Each ionizable group of an amino acid can exist in one of two states, charged or neutral. The electric charge on the functional group is determined by the relationship between its $\mathrm{p} K_{\mathrm{a}}$ and the $\mathrm{pH}$ of the solution. This relationship is described by the Henderson-Hasselbalch equation.
(a) Histidine has three ionizable functional groups. Write the equilibrium equations for its three ionizations and assign the proper $p K_{2}$ for each ionization. Draw the structure of histidine in each ionization state. What is the net charge on the histidine molecule in each ionization state?
(b) Draw the structures of the predominant ionization state of histidine at $\mathrm{pH} 1,4,8,$ and $12 .$ Note that the ionization state can be approximated by treating each ionizable group independently
(c) What is the net charge of histidine at pH $1,4,8,$ and $12 ?$ For each $\mathrm{pH}$, will histidine migrate toward the anode $(+)$ or cathode $(-)$ when placed in an electric field?

Rashmi Sinha
Rashmi Sinha
Numerade Educator
05:19

Problem 5

Mixtures of amino acids can be analyzed by first separating the mixture into its components through ionexchange chromatography. Amino acids placed on a cationexchange resin (see Fig. $3-17$ a) containing sulfonate $\left(-\mathrm{SO}_{3}^{-}\right)$ groups flow down the column at different rates because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively
charged functional groups on the amino acids, and (2) hydro-
phobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed, determine which will be eluted first from the cation-exchange column by a pH 7.0 buffer.
(a) Asp and Lys
(b) Arg and Met
(c) Glu and Val
(d) Gly and Leu
(e) Ser and Ala

KA
Kakra Atakora
Numerade Educator
01:36

Problem 6

The structure of the amino acid isoleucine is (a) How many chiral centers does it have?
(b) How many optical isomers?
(c) Draw perspective formulas for all the optical isomers of isoleucine.

Madi Sousa
Madi Sousa
Numerade Educator
05:09

Problem 7

The titration curve of alanine shows the ionization of two functional groups with $\mathrm{p} K_{\mathrm{a}}$ values of 2.34 and $9.69,$ corresponding to the ionization of the carboxyl and the protonated amino groups, respectively. The titration of di-, tri-, and larger oligopeptides of alanine also shows the ionization of only two functional groups, although the experimental $\mathrm{p} K_{\mathrm{a}}$ values are different. The trend in $\mathrm{p} K_{\mathrm{a}}$ values is summarized in the table.
(a) Draw the structure of Ala-Ala-Ala. Identify the functional groups associated with $\mathrm{p} K_{1}$ and $\mathrm{p} K_{2}$.
(b) Why does the value of $\mathrm{p} K_{1}$ increase with each additional Ala residue in the oligopeptide?
(c) Why does the value of $\mathrm{p} K_{2}$ decrease with each additional Ala residue in the oligopeptide?

Rashmi Sinha
Rashmi Sinha
Numerade Educator
01:38

Problem 8

What is the approximate molecular weight of a protein with 682 amino acid residues in a single polypeptide chain?

Ronald Prasad
Ronald Prasad
Numerade Educator
05:51

Problem 9

A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains $0.58 \%$ tryptophan $\left(M_{\mathrm{r}} 204\right)$ by weight.
(a) Calculate the minimum molecular weight of BSA (i.e., assume there is only one Trp residue per protein molecule).
(b) Size-exclusion chromatography of BSA gives a molecular weight estimate of 70,000 . How many Try residues are present in a molecule of serum albumin?

Rashmi Sinha
Rashmi Sinha
Numerade Educator
View

Problem 10

A protein has a molecular mass of 400 kDa when measured by size-exclusion chromatography. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of $180,160,$ and 60 kDa. When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands are again formed, this time with molecular masses of 160,90 , and 60 kDa. Determine the subunit composition of the protein.

Lauren Keplinger
Lauren Keplinger
Numerade Educator
07:58

Problem 11

A peptide has the sequence
$$Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly$$
(a) What is the net charge of the molecule at $\mathrm{pH} 3,8,$ and
11? (Use $\mathrm{p} K_{\mathrm{a}}$ values for side chains and terminal amino and carboxyl groups as given in Table $3-1 .$ )
(b) Estimate the pI for this peptide.

Rashmi Sinha
Rashmi Sinha
Numerade Educator
01:07

Problem 12

Pepsin is the name given to a mix of several digestive enzymes secreted (as larger precursor proteins) by glands that line the stomach. These glands also secrete hydrochloric acid, which dissolves the particulate matter in food, allowing pepsin to enzymatically cleave individual protein molecules. The resulting mixture of food, HCl, and digestive enzymes is known as chyme and has a pH near 1.5.
What pI would you predict for the pepsin proteins? What functional groups must be present to confer this pI on pepsin? Which amino acids in the proteins would contribute such groups?

Prashant Bana
Prashant Bana
Numerade Educator
02:51

Problem 13

Histones are proteins found in eukaryotic cell nuclei, tightly bound to DNA, which has many phosphate groups. The pI of histones is very high, about $10.8 .$ What amino acid residues must be present in relatively large numbers in histones? In what way do these residues contribute to the strong binding of histones to DNA?

AS
Aidan Sullivan
Numerade Educator
08:56

Problem 14

One method for separating polypeptides makes use of their different solubilities. The solubility of large polypeptides in water depends on the relative polarity of their $R$ groups, particularly on the number of ionized groups: the more ionized groups there are, the more soluble the polypeptide. Which of each pair of polypeptides that follow is more soluble at the indicated pH?
(a) (Gly) $_{20}$ or $(\mathrm{Glu})_{20}$ at $\mathrm{pH} 7.0$
(b) (Lys-Ala) $_{3}$ or (Phe-Met) $_{3}$ at pH 7.0
(c) (Ala-Ser-Gly) $_{5}$ or (Asn-Ser-His) at pH 6.0
(d) (Ala-Asp-Gly) or (Asn-Ser-His) at pH 3.0

KA
Kakra Atakora
Numerade Educator
04:07

Problem 15

A biochemist discovers and purifies a new enzyme, generating the purification table below.
(a) From the information given in the table, calculate the specific activity of the enzyme after each purification procedure.
(b) Which of the purification procedures used for this enzyme is most effective (i.e., gives the greatest relative increase in purity)?
(c) Which of the purification procedures is least effective?
(d) Is there any indication based on the results shown in the table that the enzyme after step 6 is now pure? What else could be done to estimate the purity of the enzyme preparation?

Shazia Naz
Shazia Naz
Numerade Educator
02:29

Problem 16

A purified protein is in a Hepes (N-(2-hydroxyethyl)piperazine- $N^{\prime}-(2 \text { -ethanesulfonic acid })$ ) buffer at pH 7 with $500 \mathrm{mm}$ NaCl. A sample $(1 \mathrm{mL})$ of the protein solution is placed in a tube made of dialysis membrane and dialyzed against $1 \mathrm{L}$ of the same Hepes buffer with $0 \mathrm{m}$. NaCl. Small molecules and ions (such as $\mathrm{Na}^{+}, \mathrm{Cl}^{-}$, and Hepes) can diffuse across the dialysis membrane, but the protein cannot.
(a) Once the dialysis has come to equilibrium, what is the concentration of NaCl in the protein sample? Assume no volume changes occur in the sample during the dialysis.
(b) If the original 1 mL sample were dialyzed twice, successively, against $100 \mathrm{mL}$ of the same Hepes buffer with $0 \mathrm{my} \mathrm{NaCl}$ what would be the final NaCl concentration in the sample?

David Collins
David Collins
Numerade Educator
03:59

Problem 17

At pH 7.0, in what order would the following three peptides be eluted from a column filled with a cation-exchange polymer? 'Their amino acid compositions are:
Peptide A: Ala $10 \%,$ Glu $5 \%, \operatorname{Ser} 5 \%,$ Leu $10 \%,$ Arg $10 \%$ His $5 \%,$ Ile $10 \%,$ Phe $5 \%,$ Tyr $5 \%,$ Lys $10 \%,$ Gly $10 \%,$ Pro $5 \%$ and Trp $10 \%$.
Peptide B: Ala $5 \%,$ Val $5 \%,$ Gly $10 \%,$ Asp $5 \%,$ Leu $5 \%,$ Arg $5 \%,$ Ile $5 \%,$ Phe $5 \%,$ Tyr $5 \%,$ Lys $5 \%,$ Trp $5 \%,$ Ser $5 \%,$ Thr $5 \%$ Glu $5 \%,$ Asn $5 \%,$ Pro $10 \%,$ Met $5 \%,$ and Cys $5 \%$.
Peptide C: Ala $10 \%,$ Glu $10 \%,$ Gly $5 \%,$ Leu $5 \%,$ Asp $10 \%$ Arg $5 \%,$ Met $5 \%,$ Cys $5 \%,$ Tyr $5 \%,$ Phe $5 \%,$ His $5 \%,$ Val $5 \%,$ Pro $5 \%,$ Thr $5 \%,$ Ser $5 \%,$ Asn $5 \%,$ and $\operatorname{Gln} 5 \%$.

Sana Riaz
Sana Riaz
Numerade Educator
05:22

Problem 18

A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain's own painkillers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure is consistent with each piece of information.
(a) Complete hydrolysis by 6 M HCl at $110^{\circ} \mathrm{C}$ followed by amino acid analysis indicated the presence of Gly, Leu, Phe, and $\mathrm{Tyr},$ in a 2: 1: 1: 1 molar ratio.
(b) Treatment of the peptide with 1 -fluoro- 2,4 dinitrobenzene followed by complete hydrolysis and chromatography indicated the presence of the 2,4 -dinitrophenyl derivative of tyrosine. No free tyrosine could be found.
(c) Complete digestion of the peptide with chymotrypsin followed by chromatography yielded free tyrosine and leucine, plus a tripeptide containing Phe and Gly in a 1: 2 ratio.

Rashmi Sinha
Rashmi Sinha
Numerade Educator
05:15

Problem 19

Extracts from the bacterium Bacillus brevis contain a peptide with antibiotic properties. This peptide forms complexes with metal ions and seems to disrupt ion transport across the cell membranes of other bacterial species, killing them. The structure of the peptide has been determined from the following observations.
(a) Complete acid hydrolysis of the peptide followed by amino acid analysis yielded equimolar amounts of Leu, Orn, Phe, Pro, and Val. Orn is ornithine, an amino acid not present in proteins but present in some peptides. It has the structure
(b) The molecular weight of the peptide was estimated as about 1,200.
(c) The peptide failed to undergo hydrolysis when treated with the enzyme carboxypeptidase. This enzyme catalyzes the hydrolysis of the carboxyl-terminal residue of a polypeptide
unless the residue is Pro or, for some reason, does not contain a free carboxyl group.
(d) Treatment of the intact peptide with 1 -fluoro- 2,4 dinitrobenzene, followed by complete hydrolysis and chromatography, yielded only free amino acids and the following derivative:
(Hint: The 2,4 -dinitrophenyl derivative involves the amino group of a side chain rather than the $\alpha$ -amino group.)
(e) Partial hydrolysis of the peptide followed by chromatographic separation and sequence analysis yielded the following di- and tripeptides (the amino-terminal amino acid is always at the left):
Leu-Phe Phe-Pro Orn-Leu Val-Orn
Val-Orn-Leu Phe-Pro-Val Pro-Val-Orn
Given the above information, deduce the amino acid sequence of the peptide antibiotic. Show your reasoning. When you have arrived at a structure, demonstrate that it is consistent with each experimental observation.

Sana Riaz
Sana Riaz
Numerade Educator
05:27

Problem 20

A peptide with the primary structure Lys-Arg-Pro-Leu-Ile-Asp-Gly-Ala is sequenced by the Edman procedure. If each Edman cycle is $96 \%$ efficient, what percentage of the amino acids liberated in the
fourth cycle will be leucine? Do the calculation a second time, but assume a $99 \%$ efficiency for each cycle.

Sana Riaz
Sana Riaz
Numerade Educator
01:06

Problem 21

Proteins called molecular chaperones (described in Chapter 4 ) assist in the process of protein folding. One class of chaperone found in organisms from bacteria to mammals is heat shock protein $90(\text { Hsp } 90)$. All Hsp90 chaperones contain a 10 amino acid "signature sequence," which allows for ready identification of these proteins in sequence databases. Two representations of this signature sequence are shown below.
(a) In this sequence, which amino acid residues are invariant (conserved across all species)?
(b) At which position(s) are amino acids limited to those with positively charged side chains? For each position, which amino acid is more commonly found?
(c) At which positions are substitutions restricted to amino acids with negatively charged side chains? For each position, which amino acid predominates?
(d) There is one position that can be any amino acid, although one amino acid appears much more often than any other. What position is this, and which amino acid appears
most often?

Shazia Naz
Shazia Naz
Numerade Educator
02:16

Problem 22

Three polypeptides, the sequences of which are represented below using the one-letter code for their amino acids, are present in a mixture:
1. ATKNRASCLVPKHGALMFWRHKQLVSDPILQKRQHILVCRNAAG
2. GPYFGDEPLDVHDEPEEG
3. PHLLSAWKGMEGVGKSQSFAALIVILA
Of the three, which one would migrate most slowly during chromatography through:
(a) an ion-exchange resin; beads coated with positively charged groups?
(b) an ion-exchange resin; beads coated with negatively charged groups?
(c) a size-exclusion (gel-filtration) column designed to separate small peptides such as these?
(d) Which peptide contains the ATP-binding motif shown in the following sequence logo?

David Collins
David Collins
Numerade Educator
04:41

Problem 23

Figure $3-24$ shows the amino acid sequence of bovine insulin. This structure was determined by Frederick Sanger and his coworkers. Most of this work is described in a series of articles published in the Biochemical Journal from 1945 to 1955.
When Sanger and colleagues began their work in $1945,$ it was known that insulin was a small protein consisting of two or four polypeptide chains linked by disulfide bonds. Sanger and his coworkers had developed a few simple methods for studying protein sequences.
Treatment with FDNB. FDNB (1-fluoro-2,4-dinitrobenzene) reacted with free amino (but not amido or guanidino) groups in proteins to produce dinitrophenyl (DNP) derivatives of amino acids:
Acid Hydrolysis. Boiling a protein with $10 \%$ HCl for several hours hydrolyzed all of its peptide and amide bonds. Short treatments produced short polypeptides; the longer the treatment, the more complete the breakdown of the protein into its amino acids.
Paper Chromatography. This more primitive version of thin-layer chromatography (see Fig. $10-25$ ) separated compounds based on their chemical properties, allowing identification of single amino acids and, in some cases, dipeptides. Thin-layer chromatography also separates larger peptides.
As reported in his first paper $(1945),$ Sanger reacted insulin with FDNB and hydrolyzed the resulting protein. He found many free amino acids, but only three DNP-amino acids:
$\alpha$ -DNP-glycine (DNP group attached to the $\alpha$ -amino group);$\alpha$ -DNP-phenylalanine; and $\varepsilon$ -DNP-lysine (DNP attached to the $\varepsilon \text { -amino group }) .$ Sanger interpreted these results as showing that insulin had two protein chains: one with Gly at its amino terminus and one with Phe at its amino terminus. One of the
two chains also contained a Lys residue, not at the amino terminus. He named the chain beginning with a Gly residue "A" and the chain beginning with Phe "B.". (a) Explain how Sanger's results support his conclusions.
(b) Are the results consistent with the known structure of bovine insulin (see Fig. $3-24$ )?
In a later paper $(1949),$ Sanger described how he used these techniques to determine the first few amino acids (amino-terminal end) of each insulin chain. To analyze the B chain, for example, he carried out the following steps:
1. Oxidized insulin to separate the A and B chains.
2. Prepared a sample of pure B chain with paper chromatography.
3. Reacted the B chain with FDNB.
4. Gently acid-hydrolyzed the protein so that some small peptides would be produced.
5. Separated the DNP-peptides from the peptides that did not contain DNP groups.
6. Isolated four of the DNP-peptides, which were named B1 through B4.
7. Strongly hydrolyzed each DNP-peptide to give free amino acids.
8. Identified the amino acids in each peptide with paper chromatography.
The results were as follows:
B1: $\alpha$ -DNP-phenylalanine only
B2: $\alpha$ -DNP-phenylalanine; valine
B3: aspartic acid; $\alpha$ -DNP-phenylalanine; valine
B4: aspartic acid; glutamic acid; $\alpha$ -DNP-phenylalanine; valine
(c) Based on these data, what are the first four (aminoterminal) amino acids of the $\mathrm{B}$ chain? Explain your reasoning.
(d) Does this result match the known sequence of bovine insulin (see Fig. $3-24$ )? Explain any discrepancies.
Sanger and colleagues used these and related methods to determine the entire sequence of the $A$ and $B$ chains. Their
sequence for the A chain was as follows (amino terminus on left):
Because acid hydrolysis had converted all Asn to Asp and all Gln to Glu, these residues had to be designated Asx and $\mathrm{Glx}$, respectively (exact identity in the peptide unknown). Sanger solved this problem by using protease enzymes that cleave peptide bonds, but not the amide bonds in Asn and Gln residues, to prepare short peptides. He then determined the number of amide groups present in each peptide by measuring the $\mathrm{NH}_{4}^{+}$ released when the peptide was acidhydrolyzed. Some of the results for the A chain are shown below. The peptides may not have been completely pure, so the numbers were approximate-but good enough for Sanger's purposes.
(e) Based on these data, determine the amino acid sequence of the A chain. Explain how you reached your answer. Cormpare it with Figure $3-24$.

Sana Riaz
Sana Riaz
Numerade Educator