Buffalo chymosin was isolated from abomasum mucosa extract of buffalo calves by affinity chromatography on gramicidin S-agarose followed by ion exchange chromatography on gamma-aminopropylsilochrom. Its molecular weight, 36 +/- 1 kDa, is similar to that of bovine calf chymosin. The N-terminal sequence Gly-Glu-Val-Ala-Ser-Val-Pro- coincides with that of bovine enzyme, whereas some differences were found in the amino acid composition of these enzymes. Buffalo and bovine enzyme possess similar but not identical structures. General proteolytic and milk-clotting activities of buffalo chymosin are also similar to those of bovine proteinase. pH-Optimum of its activity against hemoglobin lies at pH 4.0, somewhat higher than that for bovine chymosin, which indicates subtle differences in the functional properties of two enzymes.

The behavior of porcine pepsin in reactions of synthesis of esters and p-nitroanilides of peptides of various length has been investigated The loss of activity of the enzyme during the synthesis was found not to be connected with its inhibition, but to be due to active coprecipitation of the enzyme with the peptide forming during the synthesis. The dependence of this process on the amount of pepsin and on the length and composition of the amino and carboxyl components has been studied The pepsin appears to first enter the gel resulting from the peptide synthesis and then to transfer into the peptide precipitate. Not only pepsin, bur also nonproteolytic proteins (e.g, bovine serum albumin, lysozyme, and carbonic anhydrase) were shown to be entrapped by precipitate. Such complications may occur during the synthesis of peptides catalyzed by other proteinases.

Swine pepsin at pH 5 efficiently catalyzes a condensation between Z-Ala-Ala-Phe-OH and p-nitroanilides of Leu, Phe, Val, Ala and Arg that leads to formation of corresponding benzyloxycarbonyl-tetrapeptide p-nitroanilides with yields of 70-90%. These reactions are complicated by co-precipitation of pepsin and the reaction products that necessitates the use of a relatively high concentration of pepsin.

Calf chymosin was shown to catalyse peptide synthesis optimally over the range pH 4-5, giving satisfactory yields of methyl esters or p-nitroanilides of benzyloxycarbonyl tetra- to hexa-peptides, provided that hydrophobic amino-acid residues form the new peptide bonds. The effectiveness of the enzyme depends also on the nature of adjacent amino-acid residues. As an aspartate-proteinase with a characteristic specificity pattern chymosin would be useful for the synthesis of middle-length peptides.

Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri, tetra-, penta and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide - water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved up to 1:3.10(5) for relatively long peptides.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.

Calf chymosin catalyzes peptide synthesis optimally at pH 4-5 giving satisfactory yields of methyl esters or p-nitroanilides of benzyloxycarbonyl tetra- to hexapeptides, provided that hydrophobic amino acid residues form the new peptide bond. The enzyme efficiency depends also on the nature of adjacent amino acid residues. As an aspartyl proteinase with characteristic specificity pattern chymosin would be useful for synthesis of middle length peptides.

It has been shown that in the course of equilibrium peptide synthesis pepsin gradually disappeared from the liquid phase due to its entrapment within a gel formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin so far as inert proteins-lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin-an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.