Thermolysin Hydrolysis Research Articles

Gas chromatographic analysis of amino acids from the action of proteolytic enzymes on soil humic acids

Six proteases released more amino compounds from albumin than from a humic acid. Pronase and thermolysin released about one-sixth of the acid-hydrolysable amino acids from a humic acid, papain had slight activity while chymotrypsin, trypsin and subtilopeptidase did not attack humic acid. Thermolysin hydrolysates of four humic acids contained peptides which on acid hydrolysis broke down to increase three fold the yield of α-amino N. Net amounts of amino compounds released on incubation of thermolysin and humic acids were directly related to incubation time, substrate concentration and enzyme concentration. The amounts of each amino acid in humic acid-pronase hydrolysates exceeded those of the corresponding amino acid in pronase. By contrast the amounts of some amino acids in humic acid thermolysin hydrolysates exceeded those in the humic acid. Thus, partial autolysis of the thermolysin occurred in the presence of humic acids, despite the absence of amino acids in the enzyme control. Pronase preferentially released leucine, iso-leucine, phenylalanine and tyrosine from humic acids and albumin. The acidic amino acids, glutamic and aspartic acids, were released in poor yield from all substrates tested. The pattern of amino acids released from humic acids was controlled by the specificity of the enzyme rather than the nature and amino acid content of the substrate. However, lysine was released in small yields indicating that lysine in humic acids was somewhat protected from pronase action.

The Structure of the Bovine Pancreatic Secretory Trypsin Inhibitor—Kazal's Inhibitor: III. DETERMINATION OF THE DISULFIDE BONDS AND PROTEOLYSIS BY THERMOLYSIN

Abstract The three disulfide bonds of bovine pancreatic secretory trypsin inhibitor were assigned on the basis of the structures of cystine peptides isolated from thermolysin hydrolysates of the native inhibitor prepared at pH 6.5. The peptides were isolated by chromatographic procedures with Sephadex G-75, Sephadex G-25, and Dowex 50-X2. Five major cystine peptides were oxidized with performic acid and the cysteic acid fragments were separated by chromatography on Dowex 50-X2 and by high voltage paper electrophoresis. The cysteic acid peptides were located in the amino acid sequence of the inhibitor (Greene, L. J., and Bartelt, D. C., J. Biol. Chem., 244, 2646 (1969)) on the basis of amino acid composition and end group determination. Cystine peptides corresponding to disulfide bonds I–V, II–IV, and III–VI were isolated in 55, 44, and 91% yield, respectively. Intermediates in the degradation of the inhibitor by thermolysin having either one or two peptide bonds hydrolyzed per molecule were isolated. The sites of hydrolysis were identified as the peptide bonds Ile-Leu (residues 2 to 3) and Glu-Val (residues 12 to 13).