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A proteinase inhibitor, tentatively termed vitelloinhibitor, was purified from yolk of hen's ovarian follicles. It resembled egg-white ovoinhibitor not only in inhibitory spectrum (active for bovine trypsin and bovine chymotrypsin) but also in thermal stability, pH stability, antiserum reactivity and amino-acid composition. However, vitelloinhibitor had different molecular weight from that of ovoinhibitor. An α 2 -proteinase inhibitor preparation, isolated from laying hen's serum in the present study, was found to exhibit two bands, and the larger one of the latter corresponded to vitelloinhibitor in molecular weight. The partial N-terminal amino-acid sequence of vitelloinhibitor was the same as those of the two components of serum inhibitor and all three agreed with that of ovoinhibitor. Vitelloinhibitor is likely to be an ovoinhibitor analog derived from a serum precursor, which might be the larger component of α 2 -proteinase inhibitor.

The heat stabilization resulting from specific association of serine proteases with either of two multiheaded protease inhibitors, chicken ovoinhibitor or lima bean protease inhibitor, was determined at pH 6.7 in a differential scanning calorimeter. The 2:1 complex of either bovine alpha-chymotrypsin or subtilisin BPN' with ovoinhibitor showed two major denaturation endotherms; each 1:1 complex showed one major endotherm. Association with ovoinhibitor increased the kinetic thermal stabilities over those of the free chymotrypsin or subtilisin. Association with lima bean protease inhibitor stabilized bovine beta-trypsin greater than porcine beta-trypsin greater than bovine alpha-chymotrypsin. Complexes having different proteases bound to the same inhibitor, such as chymotrypsin . ovoinhibitor . subtilisin (1:1:1) or trypsin . inhibitor . chymotrypsin (1:1:1), denatured like mixtures of the 1:1 complexes. These results show more clearly that 2:1 association with multiheaded inhibitors stabilizes the two bound protease molecules independently. Each bound protease and the domain(s) of the inhibitor influenced by specific binding of this protease are denatured as a unit. Thus, 2:1 complexes comprise at least two new denaturing units. The extent of heat stabilization appears roughly proportional to the Kassoc determined by other methods. The results are consistent with other evidence that binding sites for proteases on multi-headed inhibitors are relatively independent in structure and function.

1. 1. Alkaline proteinases from a crude preparation of Aspergillus oryzae were isolated by one-step affinity chromatography on a chicken ovoinhibitor-Sepharose column. The yield of the activity was 55% and the purified fraction had about 300-fold higher specific activity than the starting material. 2. 2. The purified fraction was found to be homogeneous in size but consisted of at least four electrophoretically distinct bands. The amino acid composition was estimated and found to be very similar to other known alkaline proteinases from Aspergillus species, thus indicating a strong genetic relationship. 3. 3. The esterolytic activity of the purified fraction was tested on specific esteratic substrates of pancreatic serine proteases. The purified fraction was active on all 3 substrates, thus indicating a rather broad specificity and the kinetic parameters were almost identical to those of pure alkaline proteinase from Aspergillus sojae. Like the latter, the purified fraction had an extremely high kcat on acetyl tri-l-alanine methyl ester.