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
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
Effluent, mL
FIG U R E 3-6
Separation and quantitation of amino acids by ion exchange chromatography. (Courtesy of Dr. N.S. Reimer.)
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