Gary E. Means

The inhibitory properties of avian ovoinhibitors against proteolytic enzymes

Abstract 1. 1. Ovoinhibitors were isolated from egg whites of chicken, turkey, duck, penguin, and quail. Those from chicken, turkey, duck, and quail inhibited the proteolytic enzymes bovine trypsin, bovine α-chymotrypsin, subtilisin, and Aspergillus oryzae alkaline proteinase. Combining ratios of 2 trypsin and 2 α-chymotrypsin per 48 000 mol. wt. of ovoinhibitor were found. α-Chymotrypsin and subtilisin competed for the same binding areas. Sites for trypsin and α-chymotrypsin (or subtilisin) were independent and noncompetitive. The ovoinhibitor from penguin egg white inhibited trypsin, subtilisin, and A. oryzae proteinase, but not α-chymotrypsin. 2. 2. Reductive methylation of amino groups of chicken, turkey, or quail ovoinhibitors had no effect on their capacity to inhibit trypsin, α-chymotrypsin, subtilisin or A. oryzae proteinase. Modification of the same ovoinhibitors with 1,2-cyclohexane-dione, an arginine reagent, abolished their trypsin-inhibitory activity. Arginine residues were thus found essential for the trypsin-inhibitory activity of these ovoinhibitors.

An assay for carboxypeptidases A and B onpolypeptides from protein

Summary New assays for carboxypeptidase A and carboxypeptidase B were developedusing polypeptides prepared from proteins as substrates. The substrates were prepared by treating casein with either α-chymotrypsin or trypsin and then reductively methylating the amino groups of the resulting mixtures of peptides with NaBH 4 and HCHO. Reductively methylated chymotrypsin-treated casein and reductively methylated trypsin-treated casein were relatively specific substrates for carboxypeptidase A and carboxypeptidase B, respectively. Hydrolysis of COOH-terminal amino acids was quantitatively measured by reaction of newly formed amino groups with trinitrobenzenesulfonic acid. Controls containing the substrates without additions of the carboxypeptidases gave very low blank values with trinitrobenzenesulfonic acid because of the unreactivity of this reagent with dimethylamino groups. As little as 0.1 μg of either enzymes was adequate for assay. The pH optima for both enzymes were similar to the optima of these enzymes on dipeptides, but the pH profiles were broader. Carboxypeptidase B did not hydrolyze COOH-terminal e- N,N -dimethyllysines, but it rapidly hydrolyzed COOH-terminal homoarginines.