Protein Protocols Protein Protocols

Protein Hydrolysates 563
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Production of Protein Hydrolysates Using Enzymes
John M. Walker and Patricia J. Sweeney
1. Introduction
Traditionally, protein hydrolysates for amino acid analysis are produced by hydrolysis
in 6 N HCl. However, this method has the disadvantage that tryptophan is totally
destroyed, serine and threonine partially (5–10%) destroyed, and most importantly,
asparagine and glutamine are hydrolyzed to the corresponding acids. Digestion of the
protein/peptide with enzymes to produce protein hydrolysate overcomes these problems,
and is particularly useful when the concentration of asparagine and glutamine is
required. For peptides less than about 35 residues in size, complete digestion can be
achieved by digestion with aminopeptidase M and prolidase. For larger polypeptides
and proteins, an initial digestion with the nonspecific protease Pronase is required,
followed by treatment with aminopeptidase M and prolidase. Since it is important that
all enzymes have maximum activity, the following sections will discuss the general
characteristics of these enzymes.
1.1. Pronase
Pronase (EC 3.4.24.4) is the name given to a group of proteolytic enzymes that are
produced in the culture supernatant of Streptomyces griseus K-1 (1–3). Pronase is
known to contain at least ten proteolytic components: five serine-type proteases, two
Zn 2+ endopeptidases, two Zn 2+ -leucine aminopeptidases, and one Zn 2+ carboxypeptidase
(4,5). Pronase therefore has very broad specificity, hence its use in cases where
extensive or complete degradation of protein is required. The enzyme has optimal
activity at pH 7.0–8.0. However, individual components are reported to retain activity
over a much wider pH range (6–9). The neutral components are stable in the pH range
5.0–9.0 in the presence of calcium, and have optimal activity at pH 7.0–8.0. The alkaline
components are stable in the pH range 3.0–9.0 in the presence of calcium, and have
optimal activity at pH 9.0–10.0 (4). The aminopeptidase and carboxypeptidase components
are stable at pH 5.0–8.0 in the presence of calcium (9). Calcium ion dependence for
the stability of some of the components (mainly exopeptidases) was one of the earliest
observations made of Pronase (2). Pronase is therefore normally used in the presence
of 5–20 mM calcium. The addition of excess EDTA results in the irreversible loss of
70% of proteolytic activity (10). Two peptidase components are inactivated by EDTA,
but activity is restored by the addition of Co 2+ or Ca 2+ . One of these components, the
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
563