1.4 Proteins 43(1.84)The C-terminal amino acids can be removed
enzymatically by carboxypeptidase A which
preferentially cleaves amino acids with aromatic
and large aliphatic side chains, carboxypep-
tidase B which preferentially cleaves lysine,
arginine and amino acids with neutral side chains
or carboxypeptidase C which cleaves with less
specificity but cleaves proline.
1.4.1.3 PartialHydrolysis
Longer peptide chains are usually fragmented.
The fragments are then separated and analyzed
individually for amino acid sequences. Selec-
tive enzymatic cleavage of peptide bonds is
accomplished primarily with trypsin, which
cleaves exclusively Lys-X- and Arg-X-bonds,
and chymotrypsin, which cleaves peptide bonds
with less specificity (Tyr-X, Phe-X, Trp-X and
Leu-X). The enzymatic attack can be influenced
by modification of the protein. For example,
acylation of theε-amino group of lysine lim-
its tryptic hydrolysis to Arg-X (cf. 1.4.4.1.3
and 1.4.4.1.4), whereas substitution of the SH-
group of a cysteine residue with an aminoethyl
group introduces a new cleavage position
for trypsin into the molecule “pseudolysine
residue”):
(1.85)Also suited for the specific enzymatic hydrolysis
of peptide chains is the endoproteinase Glu-C
fromStaphylococcus aureusV8. It cleaves Glu-
X bonds (ammonium carbonate buffer pH 7.8or
ammonium acetate buffer pH 4.0) as well as Glu-
X plus Asp-X bonds (phosphate buffer pH 7.8).
The most important chemical method for selec-
tive cleavage uses cyanogen bromide (BrCN) to
attack Met-X-linkages (Reaction 1.86).
Hydrolysis of proteins with strong acids reveals
a difference in the rates of hydrolysis of peptide
bonds depending on the adjacent amino acid side
chain. Bonds involving amino groups of serine
and threonine are particularly susceptible to hy-
drolysis. This effect is due to