Voet D., Voet Ju.G. Biochemistry

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Problems 161

Karger,B.L., Chu,Y.-H., and Foret, F., Capillary electrophoresis of

proteins and nucleic acids, Annu. Rev. Biophys. Biomol. Struct.

24, 579–610 (1995).

Monaco, A.P. (Ed.), Pulsed Field Gel Electrophoresis. A Practical

Approach, IRL Press (1995).

Righetti, P.G., Immobilized pH gradients: Theory and methodol-

ogy, in Burdon, R.H. and van Knippenberg, P.H. (Eds.), Labo-

ratory Techniques in Biochemistry and Molecular Biology,Vol.

20, Elsevier (1990). [Discusses isoelectric focusing.]

Wehr, T., Rodríeguez-Diaz, R., and Zhu, M., Capillary Elec-

trophoresis of Proteins, Marcel Dekker (1999).

Ultracentrifugation

Graham, J., Biological Centrifugation, Bios Scientific Publishers

(2001).

Harding, S.E., Rowe, A.J., and Horton, J.C. (Eds.), Analytical Ul-

tracentrifugation in Biochemistry and Polymer Science, Royal

Society of Chemistry (1992).

Hesley, P., Defining the structure and stability of macromolecular

assemblages in solution: The re-emergence of analytical ultra-

centrifugation as a practical tool, Structure 4, 367–373 (1996).

Laue, T., Biophysical studies by ultracentrifugation, Curr. Opin.

Struct. Biol. 11, 579–583 (2001); and Laue, T.M. and Stafford,

W.F., III, Modern applications of analytical ultracentrifuga-

tion, Annu. Rev. Biophys. Biomol. Struct. 28, 75–100 (1999).

Mächtle, W. and Börger, L., Analytical Ultracentrifugation,

Springer-Verlag (2006).

Schachman, H.K., Ultracentrifugation in Biochemistry, Academic

Press (1959). [A classic treatise on ultracentrifugation.]

Schuster,T.M. and Toedt, J.M., New revolutions in the evolution of

analytical ultracentrifugation, Curr. Opin. Struct. Biol. 6,

650–658 (1996).

Stafford, W.F., III, Sedimentation velocity spins a new weave for

an old fabric, Curr. Opin. Biotech. 8, 14–24 (1997).

1. What are the ionic strengths of 1.0M solutions of NaCl,

(NH

)

, and K

? In which of these solutions would a pro-

tein be expected to be most soluble; least soluble?

2. An isotonic saline solution (one that has the same salt con-

centration as blood) is 0.9% NaCl.What is its ionic strength?

3. In what order will the following amino acids be eluted from

a column of P-cellulose ion exchange resin by a buffer at pH 6:

arginine, aspartic acid, histidine, and leucine?

4. In what order will the following proteins be eluted from a

CM-cellulose ion exchange column by an increasing salt gradient

at pH 7: fibrinogen, hemoglobin, lysozyme, pepsin, and ribonucle-

ase A (see Table 6-1)?

5. What is the order of elution of the following proteins from

a Sephadex G-50 column: catalase, ␣-chymotrypsin, concanavalin

B, lipase, and myoglobin (see Table 6-4)?

6. Estimate the molecular mass of an unknown protein that

elutes from a Sephadex G-50 column between cytochrome c and

ribonuclease A (see Table 6-4).

7. A gel chromatography column of Bio-Gel P-30 with a bed

volume of 100 mL is poured. The elution volume of the protein

hexokinase (96 kD) on this column is 34 mL.That of an unknown

protein is 50 mL.What are the void volume of the column,the vol-

ume occupied by the gel, and the relative elution volume of the

unknown protein?

8. What chromatographic method would be suitable for

separating the following pairs of substances? (a) Ala-Phe-Lys,

Ala-Ala-Lys; (b) lysozyme, ribonuclease A (see Table 6-1); and

9. What is the order of the R

values of the following amino

acids in their paper chromatography with a water/butanol/acetic

acid solvent system in which the pH of the aqueous phase is 4.5:

alanine, aspartic acid, lysine, glutamic acid, phenylalanine, and

valine?

10. What fractionation procedure could be used to purify Pro-

tein 1 from a mixture of three proteins whose amino acid compo-

sitions are as follows?

1. 25% Ala, 20% Gly, 20% Ser, 10% Ile, 10% Val, 5% Asn,

5% Gln, 5% Pro

2. 30% Gln, 25% Glu, 20% Lys, 15% Ser, 10% Cys

3. 25% Asn, 20% Gly, 20% Asp, 20% Ser, 10% Lys, 5% Tyr

All three proteins are similar in size and pI, and there is no anti-

body available for Protein 1.

*11. Purification tables are often used to keep track of the

yield and purification of a protein.The specific activity is the ratio

of the amount of the protein of interest, here myoglobin (Mb), ob-

tained at given step (in ␮mol or enzyme units) divided by the

amount (mg) of total protein. The yield is the ratio of the amount

of the protein of interest obtained at a given purification step (in

␮mol or enzyme units) divided by the original amount present in

the crude extract, often converted to percent yield by multiplying

by 100. The fold purification is the ratio of the specific activity of

the purified protein to that of the crude extract.

(a) For the purification table below, calculate the specific ac-

tivity, % yield, and fold purification for the empty cells.

(b) Which step, DEAE or affinity chromatography, causes the

greatest loss of Mb?

(d) If you could use only one purification step, which tech-

nique would be best?

PROBLEMS

mg Total ␮mol Specific Activity Fold

Purification Step Protein Mb (␮mol protein/mg total protein) % Yield Purification

1. Crude extract 1550 0.75 100 1

2. DEAE-cellulose chromatography 550 0.35

3.Affinity chromatography 5.0 0.28

Purification Table (for Problem 11)

JWCL281_c06_129-162.qxd 5/31/10 2:10 PM Page 161

162 Chapter 6. Techniques of Protein and Nucleic Acid Purification

12. The neurotransmitter ␥-aminobutyric acid is thought to

bind to specific receptor proteins in nerve tissue. Design a proce-

dure for the partial purification of such a receptor protein.

13. A mixture of amino acids consisting of arginine, cysteine,

glutamic acid, histidine, leucine, and serine is applied to a strip of

paper and subjected to electrophoresis using a buffer at pH 7.5.

What are the directions of migration of these amino acids and

what are their relative mobilities?

*14. Sketch the appearance of a fingerprint of the following

tripeptides: Asn-Arg-Lys, Asn-Leu-Phe, Asn-His-Phe, Asp-Leu-

Phe, and Val-Leu-Phe.Assume the paper chromatographic step is

carried out using a water/butanol/acetic acid solvent system (pH 4.5)

and the electrophoretic step takes place in a buffer at pH 6.5.

15. What is the molecular mass of a protein that has a relative

electrophoretic mobility of 0.5 in an SDS–polyacrylamide gel such

as that of Fig. 6-25?

16. Explain why the molecular mass of fibrinogen is signifi-

cantly overestimated when measured using a calibrated gel filtra-

tion column (Fig. 6-10) but can be determined with reasonable

accuracy from its electrophoretic mobility on an SDS–polyacryl-

amide gel (see Table 6-4).

17. (a) What would be the relative arrangement of the follow-

ing proteins after they had been subjected to isoelectric focusing:

insulin, cytochrome c, histone, myoglobin, and ribonuclease A?

(b) Sketch the appearance of a two-dimensional gel electrophore-

togram of cytochrome c, myoglobin, and ribonuclease A (see

Tables 6-1 and 6-4).

18. Calculate the centrifugal acceleration, in gravities (g’s), on

a particle located 6.5 cm from the axis of rotation of an ultracen-

trifuge rotating at 60,000 rpm (1 g ⫽ 9.81 m ⴢ s

–2

19. In a dilute buffer solution at 20°C, rabbit muscle aldolase

has a frictional coefficient of 8.74 ⫻ 10

–8

g ⴢ s

–1

, a sedimentation co-

efficient of 7.35 S, and a partial specific volume of 0.742 cm

ⴢ g

–1

Calculate the molecular mass of aldolase assuming the density of

the solution to be 0.998 g ⴢ cm

–3

*20. The sedimentation coefficient of a protein was measured

by observing its sedimentation at 20°C in an ultracentrifuge spin-

ning at 35,000 rpm.

Time, t Distance of Boundary from

(min) Center of Rotation, r (cm)

4 5.944

6 5.966

8 5.987

10 6.009

12 6.032

The density of the solution is 1.030 g ⴢ cm

–3

, the partial specific vol-

ume of the protein is 0.725 cm

ⴢ g

–1

, and its frictional coefficient is

3.72 ⫻ 10

–8

g ⴢ s

–1

. Calculate the protein’s sedimentation coeffi-

cient, in svedbergs, and its molecular mass.

JWCL281_c06_129-162.qxd 2/22/10 2:26 PM Page 162

GG G G GGG

200 210 220 230 240

AAA A AA AAAAAA AACCCCCCCCCCCCCCCTTT TTTTT T TT T

GGGGGGG N

120 130 140 1501100

GG GG G G GAAA A AA ACCCCCCCCC CC CCCCCTT T T T TTTT TT TTT

163

CHAPTER 7

Covalent Structures

of Proteins and

Nucleic Acids

1 Primary Structure Determination of Proteins

A. End Group Analysis: How Many Different Types of Subunits?

B. Cleavage of the Disulfide Bonds

C. Separation, Purification, and Characterization of the

Polypeptide Chains

D. Specific Peptide Cleavage Reactions

E. Separation and Purification of the Peptide Fragments

F. Sequence Determination

G. Ordering the Peptide Fragments

H. Assignment of Disulfide Bond Positions

I. Peptide Characterization and Sequencing by Mass

Spectrometry

J. Peptide Mapping

2 Nucleic Acid Sequencing

A. The Sanger Method

B. Genome Sequencing

C. Next Generation DNA Sequencing Technologies

D. Nucleic Acid Sequencing versus Amino Acid Sequencing

3 Chemical Evolution

A. Sickle-Cell Anemia: The Influence of Natural Selection

B. Species Variations in Homologous Proteins: The Effects of

Neutral Drift

C. Evolution through Gene Duplication

4 Bioinformatics: An Introduction

A. Sequence Databases

B. Sequence Alignment

C. Construction of Phylogenetic Trees

5 Chemical Synthesis of Polypeptides

A. Synthetic Procedures

B. Problems and Prospects

6 Chemical Synthesis of Oligonucleotides

A. Synthetic Procedures

B. DNA Microarrays

C. SELEX

many other molecules. In the form of muscle fibers and

other contractile assemblies, proteins generate the coor-

dinated mechanical motion of numerous biological

processes, such as the separation of chromosomes during

cell division and the movement of your eyes as you read

this page. Proteins, such as rhodopsin in the retina of your

eye, acquire sensory information that is processed

through the actions of nerve cell proteins. The proteins of

the immune system, such as the immunoglobulins, form

an essential biological defense system in higher animals.

Proteins are major active elements in, as well as products

of, the expression of genetic information. However, pro-

teins also have important passive roles, such as that of col-

lagen, which provides bones, tendons, and ligaments with

their characteristic tensile strength. Clearly, there is con-

siderable validity to the old cliché that proteins are the

“building blocks” of life.

The function of DNA as the genetic archive and the as-

sociation of RNA with protein synthesis have been known

since the mid-twentieth century. However, it was not until

the 1970s that it became clear that RNA can form struc-

tures whose complexities rival those of proteins, and it was

not until the mid-1980s that it was shown that RNA has

biologically important catalytic functions.

Protein and nucleic acid function can best be under-

stood in terms of their structures, that is, the three-dimen-

sional relationships between their component atoms. The

structural descriptions of proteins and nucleic acids, as

well as those of other polymeric materials, have been tra-

ditionally described in terms of four levels of organization

(Fig. 7-1):

1. Primary structure (1° structure), which for a protein

is the amino acid sequence of its polypeptide chain(s) and

for a nucleic acid is its base sequence.

2. Secondary (2°) structure, which is the local spatial

arrangement of a polypeptide’s or a nucleic acid’s back-

bone atoms without regard to the conformations of their

side chains or bases.

3. Tertiary (3°) structure, which refers to the three-

dimensional structure of an entire polypeptide or polynu-

cleotide chain. The distinction between secondary and ter-

tiary structures is, of necessity, somewhat vague; in practice,

the term “secondary structure” alludes to easily character-

ized structural entities such as helices.

Proteins are at the center of the action in biological

processes. They function as enzymes, which catalyze the

complex set of chemical reactions that are collectively re-

ferred to as life. Proteins serve as regulators of these reac-

tions, both directly as components of enzymes and indi-

rectly in the form of chemical messengers, known as

hormones, as well as the receptors for those hormones.

They act to transport and store biologically important

substances such as metal ions, O

, glucose, lipids, and

JWCL281_c07_163-220.qxd 6/1/10 7:03 AM Page 163

4. Most proteins are composed of two or more polypep-

tide chains, loosely referred to as subunits, which associate

through noncovalent interactions and, in some cases, disul-

fide bonds. A protein’s quaternary (4°) structure refers to

the spatial arrangement of its subunits. A nucleic acid’s

quaternary structure is similarly defined.

In this chapter, we discuss the 1° structures of proteins

and nucleic acids: how they are elucidated and their bio-

logical and evolutionary significance. We also survey the

field of bioinformatics as well as methods of chemically

synthesizing polypeptide and oligonucleotide chains. The

2°, 3°, and 4° structures of proteins and nucleic acids, as we

shall see, are a consequence of their 1° structures. For pro-

teins, these topics are treated in Chapters 8 and 9, whereas

for nucleic acids, they are discussed mainly in Chapters 29,

31, and 32.

1 PRIMARY STRUCTURE

DETERMINATION OF PROTEINS

See Guided Exploration 4: Protein sequence determination The

first determination of the complete amino acid sequence of

a protein, that of the bovine polypeptide hormone insulin

by Frederick Sanger in 1953, was of enormous biochemical

significance in that it definitively established that proteins

have unique covalent structures. Since that time, the amino

acid sequences of tens of thousands of proteins have been

elucidated. This extensive information has been of central

importance in the formulation of modern concepts of bio-

chemistry for several reasons:

1. The knowledge of a protein’s amino acid sequence is

essential for an understanding of its molecular mechanism

of action as well as being prerequisite for the elucidation

of its three-dimensional structure by both X-ray crystal-

lography and nuclear magnetic resonance (NMR) spec-

troscopy (Section 8-3A).

2. Sequence comparisons among analogous proteins

from the same individual, from members of the same

species, and from members of related species have yielded

important insights into how proteins function and have indi-

cated the evolutionary relationships among the proteins and

the organisms that produce them.These analyses, as we shall

see in Section 7-3, complement and extend corresponding

taxonometric studies based on anatomical comparisons.

3. Amino acid sequence analyses have important clini-

cal applications because many inherited diseases are

caused by mutations leading to an amino acid change in a

protein. Recognition of this fact has led to the develop-

ment of valuable diagnostic tests for many such diseases

and, in many cases, to symptom-relieving therapy.

The elucidation of the 51-residue primary structure of

insulin (Fig. 7-2) was the labor of many scientists over a

164 Chapter 7. Covalent Structures of Proteins and Nucleic Acids

Secondary

structure

(helix)

Primary structure (amino acid sequence in a polypeptide chain)

Quaternary structure:

the four separate chains

of hemoglobin assembled

into an oligomeric protein

Tertiary structure:

one complete protein chain

( chain of hemoglobin)

(b)

(a)

– Lys – Ala – His – Gly – Lys – Lys – Val – Leu – Gly - Ala –

Figure 7-1 The structural hierarchy in proteins. (a) Primary structure, (b) secondary structure,

Irving Geis Collection, Howard Hughes Medical Institute. Reprinted with permission.]

JWCL281_c07_163-220.qxd 8/10/10 12:36 AM Page 164

period of a decade that altogether utilized ⬃100 g of pro-

tein. Procedures for primary structure determination have

since been so refined and automated that proteins of simi-

lar size can be sequenced by an experienced technician in a

few days using only a few micrograms of protein. The se-

quencing of the 1021-residue enzyme ␤-galactosidase in

1978 signaled that the sequence analysis of almost any pro-

tein could be reasonably attempted. Despite these techni-

cal advances, the basic procedure for primary structure de-

termination using the techniques of protein chemistry is

that developed by Sanger.The procedure consists of three

conceptual parts, each of which requires several laboratory

steps:

1. Prepare the protein for sequencing:

a. Determine the number of chemically different

polypeptide chains (subunits) in the protein.

b. Cleave the protein’s disulfide bonds.

c. Separate and purify the unique subunits.

2. Sequence the polypeptide chains:

a. Fragment the individual subunits at specific points

to yield peptides small enough to be sequenced di-

rectly.

b. Separate and purify the fragments.

c. Determine the amino acid sequence of each pep-

tide fragment.

d. Repeat Step 2a with a fragmentation process of

different specificity so that the subunit is cleaved

at peptide bonds different from before. Separate

these peptide fragments as in Step 2b and deter-

mine their amino acid sequences as in Step 2c.

3. Organize the completed structure:

a. Span the cleavage points between one set of pep-

tide fragments by the other. By comparison, the

sequences of these sets of polypeptides can be

arranged in the order that they occur in the sub-

unit, thereby establishing its amino acid sequence.

b. Elucidate the positions of the disulfide bonds, if

any, between and within the subunits.

We discuss these various steps in the following sections.

A. End Group Analysis: How Many Different Types

of Subunits?

Each polypeptide chain (if it is not chemically blocked or

circular) has an N-terminal residue and a C-terminal

residue. By identifying these end groups, we can establish

the number of chemically distinct polypeptides in a protein.

For example, insulin has equal amounts of the N-terminal

residues Phe and Gly, which indicates that it has equal

numbers of two nonidentical polypeptide chains.

a. N-Terminus Identification

There are several effective methods by which a polypep-

tide’s N-terminal residue may be identified. 1-Dimethyl-

aminonaphthalene-5-sulfonyl chloride (dansyl chloride)

reacts with primary amines (including the ε-amino group of

Lys) to yield dansylated polypeptides (Fig. 7-3). Acid hy-

drolysis liberates the N-terminal residue as a dansylamino

acid, which exhibits such intense yellow fluorescence that it

can be chromatographically identified from as little as 100

picomoles of material [1 picomole (pmol)  10

12

mol].

In the most useful method of N-terminal residue identi-

fication, the Edman degradation (named after its inventor,

Pehr Edman), phenylisothiocyanate (PITC, Edman’s

reagent) reacts with the N-terminal amino groups of pro-

teins under mildly alkaline conditions to form their

phenylthiocarbamyl (PTC) adduct (Fig. 7-4). This product

is treated with an anhydrous strong acid such as trifluo-

roacetic acid, which cleaves the N-terminal residue as its

thiazolinone derivative but does not hydrolyze other pep-

tide bonds. The Edman degradation therefore releases the

N-terminal amino acid residue but leaves intact the rest of

the polypeptide chain.The thiazolinone-amino acid is selec-

tively extracted into an organic solvent and is converted to

the more stable phenylthiohydantoin (PTH) derivative by

treatment with aqueous acid.This PTH-amino acid is most

commonly identified by comparing its retention time on

HPLC with those of known PTH-amino acids.

The most important difference between the Edman

degradation and other methods of N-terminal residue

identification is that we can determine the amino acid se-

quence of a polypeptide chain from the N-terminus inward

by subjecting the polypeptide to repeated cycles of the Ed-

man degradation and, after every cycle,identifying the newly

liberated PTH-amino acid. This technique has been auto-

mated, resulting in great savings of time and materials

(Section 7-1F).

b. C-Terminus Identification

There is no reliable chemical procedure comparable to

the Edman degradation for the sequential end group analy-

sis from the C-terminus of a polypeptide. This can be done

enzymatically, however, using exopeptidases (enzymes that

Section 7-1. Primary Structure Determination of Proteins 165

Figure 7-2 Primary structure of bovine insulin. Note the intrachain and interchain disulfide

bond linkages.

Ala Tyr

Asn

Gly

Asn

GlnGlu Leu

Ile

Val Cys CysSer Val Cys Ser Glu

2110

Tyr Gln Leu

Cys

CysLeu

His

GlnAsnValPhe

S S

Gly

HisSer Leu Val AlaGlu

Leu

Tyr

Leu Val Cys

1510

Gly Glu

Arg

Gly

Phe Phe

Tyr

Thr

Pro Lys

Ala

B chain

chain

JWCL281_c07_163-220.qxd 2/22/10 9:10 PM Page 165

cleave a terminal residue from a polypeptide). One class of

exopeptidases, the carboxypeptidases, catalyzes the hy-

drolysis of the C-terminal residues of polypeptides:

Carboxypeptidases, like all enzymes, are highly specific (se-

lective) for the chemical identities of the substances whose

reactions they catalyze (Section 13-2). The side chain spe-

cificities of the various carboxypeptidases in common use

are listed in Table 7-1. The second type of exopeptidase

NH C

...

n–2

NH CCH

n–1

NH C

...

n–2

COO

–

NH CH

n–1

NH CH

COO

–

carboxypeptidase

COO

–

listed in Table 7-1, the aminopeptidases, sequentially

cleave amino acids from the N-terminus of a polypeptide

and have been similarly used to determine N-terminal se-

quences.

Why can’t carboxypeptidases be used to determine

amino acid sequences? If a carboxypeptidase cleaved all

C-terminal residues at the same rate, irrespective of their

identities, then by following the course of appearance of the

various free amino acids in the reaction mixture (Fig. 7-5a),

the sequence of several amino acids at the C-terminus

could be determined. If, however, the second amino acid

residue, for example, were cleaved at a much faster rate

than the first, both amino acids would appear to be re-

leased simultaneously (Fig. 7-5b). Carboxypeptidases, in

fact, exhibit selectivity toward side chains, so their use, ei-

ther singly or in mixtures, rarely reveals the order of more

than the first few C-terminal residues of a polypeptide.

C-Terminal residues with a preceding Pro residue are

not subject to cleavage by carboxypeptidases A and B

(Table 7-1). Chemical methods are therefore usually em-

ployed to identify their C-terminal residue. In the most re-

liable such chemical method, hydrazinolysis, a polypeptide

is treated with anhydrous hydrazine at 90°C for 20 to 100 h

166 Chapter 7. Covalent Structures of Proteins and Nucleic Acids

Figure 7-3 The reaction of dansyl chloride in end group analysis.

NH CCH

COOHCH

NH CCH

Polypeptide1-Dimethylaminonaphthalene-

5-sulfonyl chloride (dansyl chloride)

–

HCl

NH CCH

CCH

Dansyl polypeptide

...

Dansylamino acid

(fluorescent)

...

OH +

Free amino acids

...

N(CH

)

N(CH

)

N(CH

)

OSO

COOH

JWCL281_c07_163-220.qxd 2/22/10 9:11 PM Page 166

Section 7-1. Primary Structure Determination of Proteins 167

Figure 7-4 The Edman degradation. Note that the reaction occurs in three separate

stages that each require quite different conditions.Amino acid residues can therefore be

sequentially removed from the N-terminus of a polypeptide in a controlled stepwise

fashion.

See the Animated Figures

Figure 7-5 Hypothetical rate of the carboxypeptidase-catalyzed

release of amino acids from peptides having the indicated

C-terminal sequences. (a) All bonds cleaved at the same rate.

(b) Ser removed slowly, Tyr cleaved rapidly, and Leu cleaved at

an intermediate rate.

NH CCH

PolypeptidePhenylisothiocyanate

(PITC)

anhydrous

CCOOH

CCH

NH CCH

PTC polypeptide

...

HC C

–

CCH

NH CCH

Original polypeptide less

its N-terminal residue

Thiazolinone derivative

HC C

NCS

PTH-amino acid

...

1.0

Time

Ser

Tyr

Leu

Ser

Tyr

Leu

(a) Starting peptide: –Ser–Leu–Tyr

(b) Starting peptide: –Leu–Tyr–Ser

Molar equivalents of amino acid released

JWCL281_c07_163-220.qxd 2/22/10 9:11 PM Page 167

in the presence of a mildly acidic ion exchange resin (which

acts as a catalyst):

All the peptide bonds are thereby cleaved, yielding the

aminoacyl hydrazides of all the amino acid residues except

that of the C-terminal residue, which is released as the free

amino acid and therefore can be identified chromato-

graphically. Unfortunately, hydrazinolysis is subject to a

great many side reactions that have largely limited its ap-

plication to carboxypeptidase-resistant polypeptides.

B. Cleavage of the Disulfide Bonds

The next step in the sequence analysis is to cleave the disul-

fide bonds between Cys residues.This is done for two reasons:

1. To permit the separation of polypeptide chains (if

they are disulfide linked).

CCH

O O

...

Polypeptide

Free amino acid

Hydrazine

acidic ion exchange

resin catalyst

Aminoacyl

hydrazides



n1

NH CCH

NH COO



CCH



COO





NH NH

...



n1

CCH

2. To prevent the native protein conformation, which is

stabilized by disulfide bonds, from obstructing the action of

the proteolytic (protein-cleaving) agents used in primary

structure determinations (Section 7-1D).

Disulfide bond locations are established in the final step of

the sequence analysis (Section 7-1H).

Disulfide bonds are most often cleaved reductively by

treatment with sulfhydryl-containing compounds such as

2-mercaptoethanol:

In order to expose all disulfide groups to the reducing

agent, the reaction is usually carried out under conditions

that denature the protein. The resulting free sulfhydryl

groups are alkylated, usually by treatment with iodoacetic

acid, to prevent the reformation of disulfide bonds through

NH C

...

NH C

...

Cystine

Cysteine

2-Mercaptoethanol

...

2 HSCH

NH C

...

NH C

...

SCH

168 Chapter 7. Covalent Structures of Proteins and Nucleic Acids

Table 7-1 Specificities of Various Exopeptidases

Enzyme Source Specificity

Carboxypeptidase A Bovine pancreas R

Arg, Lys, Pro; R

n1

Pro

Carboxypeptidase B Bovine pancreas R

Arg, Lys, R

n1

Pro

Carboxypeptidase C Citrus leaves All free C-terminal residues; pH optimum  3.5

Carboxypeptidase Y Yeast All free C-terminal residues, but slowly with R

 Gly

Leucine aminopeptidase Porcine kidney R

Pro

Aminopeptidase M Porcine kidney All free N-terminal residues





 the N-terminal residue; R

 the C-terminal residue.

JWCL281_c07_163-220.qxd 2/22/10 9:11 PM Page 168

oxidation by O

. S-Alkyl derivatives are stable in air and

under the conditions used for the subsequent cleavage of

peptide bonds.

C. Separation, Purification, and Characterization of

the Polypeptide Chains

A protein’s nonidentical polypeptides must be separated

and purified in preparation for their amino acid sequence

determination. Subunit dissociation, as well as denatura-

tion, occurs under acidic or basic conditions, at low salt

concentrations, at elevated temperatures, or through the

use of denaturing agents such as urea, guanidinium ion

(Section 5-5G), or detergents such as sodium dodecyl sul-

fate (SDS; Section 6-4C).The dissociated subunits can then

be separated by methods described in Chapter 6 that capi-

talize on small differences in polypeptide size and polarity.

Ion exchange and gel filtration chromatography, usually by

HPLC (Section 6-3Dg), are most often used.

It is, of course,desirable to know the number of residues

in the polypeptide to be sequenced, which can be estimated

from its molecular mass (⬃110 D/residue). Molecular mass

can be measured with an accuracy of no better than 5 to

Cys

Iodoacetate

S-Carboxymethylcysteine

COO

–

COO

–

Cysteine

+ HI

10% by the more traditional laboratory techniques of gel

filtration chromatography and SDS–PAGE (Sections 6-3Ba

and 6-4C). In recent years, however, mass spectrometry

(Section 7-1I) has provided a faster and far more accurate

means to determine the molecular masses of macromole-

cules. Mass spectrometry can determine the molecular

masses of picomolar amounts of 100 kD polypeptides

with accuracies of ⬃0.01%.

D. Specific Peptide Cleavage Reactions

Polypeptides that are longer than 40 to 100 residues cannot

be directly sequenced (Section 7-1F). Polypeptides of greater

length must therefore be cleaved, either enzymatically or

chemically, to fragments small enough to be sequenced (the

polypeptides produced by E. coli and humans have broad

distributions of lengths that average ⬃320 and ⬃470

residues, respectively). In either case, the cleavage process

must be complete and highly specific so that the aggregate

sequence of a subunit’s peptide fragments, when correctly

ordered, is that of the intact subunit.

a. Trypsin Specifically Cleaves Peptide Bonds after

Positively Charged Residues

Endopeptidases (enzymes that catalyze the hydrolysis

of internal peptide bonds), like exopeptidases, have side

chain requirements for the residues flanking the scissile

(to be cleaved) peptide bond. The side chain specificities

of the endopeptidases most commonly used to fragment

polypeptides are listed in Table 7-2.The digestive enzyme

trypsin has the greatest specificity and is therefore the

Section 7-1. Primary Structure Determination of Proteins 169

Table 7-2 Specificities of Various Endopeptidases

Enzyme Source Specificity Comments

Trypsin Bovine pancreas R

n–1

 positively charged Highly specific

residues:Arg, Lys; R

Pro

Chymotrypsin Bovine pancreas R

n–1

 bulky hydrophobic residues: Cleaves more slowly for

Phe,Trp,Tyr;R

Pro R

n–1

 Asn, His, Met, Leu

Elastase Bovine pancreas R

n–1

 small neutral residues:

Ala, Gly, Ser,Val; R

Pro

Thermolysin Bacillus thermoproteolyticus R

 Ile, Met, Phe,Trp,Tyr, Val; Occasionally cleaves at R

 Ala,

n–1

Pro Asp, His, Thr; heat stable

Pepsin Bovine gastric mucosa R

 Leu, Phe,Trp,Tyr; Also others; quite nonspecific;

n–1

Pro pH optimum 2

Endopeptidase Arg-C Mouse submaxillary gland R

n–1

 Arg May cleave at R

n–1

 Lys

Endopeptidase Asp-N Pseudomonas fragi R

 Asp May cleave at R

 Glu

Endopeptidase Glu-C Staphylococcus aureus R

n–1

 Glu May cleave at R

n–1

 Gly

Endopeptidase Lys-C Lysobacter enzymogenes R

n–1

 Lys May cleave at R

n–1

 Asn



NH CH C

n1

Scissile

peptide bond

JWCL281_c07_163-220.qxd 2/22/10 9:11 PM Page 169

most valuable member of the arsenal of peptidases used

to fragment polypeptides. It cleaves peptide bonds on the

C-side (toward the carboxyl terminus) of the positively

charged residues Arg and Lys if the next residue is not

Pro:

The other endopeptidases listed in Table 7-2 exhibit

broader side chain specificities than trypsin and often yield

a series of peptide fragments with overlapping sequences.

However, through limited proteolysis, that is, by adjusting

reaction conditions and limiting reaction times, these less

specific endopeptidases can yield useful peptide fragments.

This is because the complex native structure of a protein

(subunit) buries many otherwise enzymatically susceptible

peptide bonds beneath the surface of the protein molecule.

With proper conditions and reaction times, only those pep-

tide bonds in the native protein that are initially accessible

to the peptidase will be hydrolyzed. Limited proteolysis is

often employed to generate peptide fragments of useful

size from subunits that have too many or too few Arg and

Lys residues to do so with trypsin (although if too many are

present, limited proteolysis with trypsin may also yield use-

ful fragments).

b. Cyanogen Bromide Specifically Cleaves Peptide

Bonds after Met Residues

Several chemical reagents promote peptide bond cleav-

age at specific residues. The most useful of these, cyanogen

bromide (CNBr), causes specific and quantitative cleavage

NH C

...

NH CCH

...

Any amino acid

residue but Pro



NH C

...



NCCH

...

trypsin

Lys

(or Arg)

on the C-side of Met residues to form a peptidyl homoser-

ine lactone:

The reaction is performed in an acidic solvent (0.1M HCl

or 70% formic acid) that denatures most proteins so that

cleavage normally occurs at all Met residues.



Cyanogen

bromide

Methyl

thiocyanate

Peptidyl

homoserine

lactone

Aminoacyl

peptide



...

NH CH

...

NH CH

...

NCH

...



S NC

170 Chapter 7. Covalent Structures of Proteins and Nucleic Acids

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