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chapter 3

Protein Isolation and Determination of Amino Acid Sequence

3.5 Amino Acid Composition

Determination of the amino acid sequence of a purified

protein begins with determination of the amino acid com-

position, i.e., the number of moles of each amino acid

residue present per mole of protein, as follows.

The protein is completely hydrolyzed by acid

( 6

N HC1,

24 hours or longer at 110°C, under vacuum or inert gas) to

its constituent amino acids and the resultant hydrolysate is

evaporated to dryness. The amino acid composition is de-

termined on protein hydrolysates obtained after 24,48, and

72 hours of acid treatment. The content of amino acids with

bulky aliphatic side chains such as isoleucine, leucine, and

valine, which undergo slow hydrolysis, is calculated from

an extrapolation of the hydrolysate data to infinite time.

The content of hydroxyl-containing amino acids, which

are slowly destroyed during hydrolysis, is obtained by a

corresponding extrapolation to zero time. Since cysteine,

cystine, and methionine residues are somewhat unstable

to hydrolysis, these residues are oxidized to cysteic acid

and methionine sulfone, respectively, with performic acid

before quantitative analysis. Cysteine, or half-cystine, is

quantitated as a derivative such as carboxymethyl cys-

teine after reduction and alkylation, a necessary pre-

requisite to subsequent sequence analysis. Tryptophan

released on mixing and to allow time for the formation of

precipitates.

loss due to oxidation during hydrolysis can be greatly

reduced by the inclusion of reducing agents—usually low-

molecular-weight thiols—or oxygen-halide scavengers in

the acid hydrolysis procedure. Tryptophan is substantially

preserved if hydrolysis is performed with strong acids that

do not contain halogen (e.g., methane sulfonic acid) or

with strong alkali (e.g., NaOH). During acid hydrolysis,

asparagine (Asn) and glutamine (Gin) are hydrolyzed to

aspartic acid (Asp) and glutamic acid (Glu), respectively,

and NH3. Thus, Asn and Gin do not appear in the elution

profile, and the Asp and Glu quantities include Asn and

Gin, respectively.

The hydrolysate is dissolved in a small volume of an

acidic buffer to obtain the protonated form of the amino

acid and then chromatographed on a cation exchange resin

column, e.g., Dowex 50. The SO^" groups of the resin bind

all the protonated amino acids at the top of the column.

The intensity of binding of each type of amino acid to the

resin depends upon the number of positive charges on the

molecules, the nature and size of the R-groups, and the pK'

values of the functional groups of the amino acids. Basic

amino acids (lysine, histidine and arginine) have more than

one positive charge and therefore bind more tightly than

the neutral amino acids. Acidic amino acids (glutamic and

aspartic acid) bind less tightly.

Amino acids are weakly attracted by hydrophobic and

van der Waals interactions through their side chains. The

strength of these attractions is determined by the adsorp-

tion characteristics of the resin and leads to different

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40

Effluent, mL

FIG U R E 3-6

Separation and quantitation of amino acids by ion exchange chromatography. (Courtesy of Dr. N.S. Reimer.)