Search

From the chymotryptic hydrolysate of 1.08 g of the precipitate obtained on treatment of alkaline protease from Aspergillus flavus with trichloracetic acid 134 peptides were isolated by means of ion exchange chromatography, paper electrophoresis and paper chromatography. Among these 38 peptides containing 311 amino acids were isolated in amounts exceeding 0.50μmol. The peptides were characterized by amino acid analysis, electric charge and also mostly by the terminal groups determination. In the case of peptides isolated in larger amounts the complete or at least the partial sequence of amino acids has been determined. In all peptides the total isolated amount in μmol was determined. The peptides containing basic amino acids were subfractionated with trypsin.

A partly autolyzed preparation of the alkaline protease from As. flavus, denatured by trichloroacetic acid, was resolved into the high and low molecular weight part; the amino acid composition of both parts was similar to that of the native protease. The high molecular weight part was subjected to tryptic digestion; the digest was fractionated by chromatography on a sulfonated ion exchange resin and by paper electrophoresis and paper chromatography techniques. The peptides isolated were characterized by amino acid analysis and terminal end group analysis. Peptides obtained in larger quantities were sequenced. The data on peptides obtained in this study are compared with the results of earlier work on nonradioactive peptides from a partial hydrolysate of the [32P]DIP-labeled enzyme.

1. 1. From the soluble portion of the tryptic digest of oxidized diisopropyl [32P]-phosphoryl-derivative of alkaline proteinase of Aspergillus flavus a radioactive fragment was isolated and its amino acid composition determined to be Asp7, Met1, Thr5, Ser8, Glu4, Pro3, Gly7, Ala9, Val6, Ile5, Leu6, Tyr2 and Phe2. 2. 2. Four radioactive peptides were isolated from a partial acid hydrolysate of the slightly soluble portion of the tryptic digest. These peptides provides evidence of the amino acid sequence Gly-Thr-Ser-Met-Ala around the reactive serine residue of alkaline proteinase. This amino acid sequence represents a part of the large tryptic fragment. 3. 3. Similarities in the amino acid sequences of serine-containing active centers of microbial proteinases are discussed.