The Proteolytic Enzymes of the K-1 Strain of Streptomyces griseus Obtained from a Commercial Preparation (Pronase)

Abstract A study was carried out on some of the properties of the two smallest serine endopeptidases from Pronase which had previously been purified to homogeneity. Each enzyme is homologous with bovine chymotrypsin and has isoleucine as the NH2-terminal residue. The smaller enzyme is free of lysine, whereas the larger enzyme contains only 1 lysine residue. Reaction of the larger enzyme with acetic anhydride yielded a homogeneous, active, and stable derivative as indicated by ion exchange chromatography and acrylamide gel electrophoresis. Reaction of the smaller enzyme with acetic anhydride yielded two chromatographic components, of which only the larger demonstrated activity against Nα-acetyl-l-tyrosine ethyl ester. This active component autolyzes during acrylamide gel electrophoresis but appears as a single band by cellulose acetate electrophoresis. The excellent yields of these protein derivatives were only achieved by modifying the past standard techniques of acetylation. In each reaction mixture glycerol was included at a concentration of 20% by volume. As a result only small amounts of native proteins were required to prepare the derivatives. Analysis of each enzyme revealed complete acetylation of the NH2-terminal isoleucine residue. Acetylation resulted in only modest changes in the Michaelis constant and maximal velocity of each enzyme with Nα-acetyl-l-tyrosine ethyl ester as substrate. Despite the earlier observation that only the larger enzyme demonstrated marked stability in 6 m guanidinium chloride there was no difference in the heat stabilities of the two enzymes. Neither showed an effect of ethylenediaminetetraacetate (EDTA) on activity at 37° even after several hours; however, at temperatures above 45° each enzyme underwent marked loss of activity in the presence of EDTA, whereas activity was conserved up to 60° in the absence of chelating agent. Metal-free enzyme was prepared in each case by gel filtration at a low pH in the absence of metals. These metal-free proteins demonstrated the same temperature stabilities as the EDTA-treated native enzymes. Of the many cations tested, Ca2+ was specific in each case in restoring the stability at higher temperatures to the metal-free enzymes.