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The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

Three aspartic proteinases with similar Mr values (approx. 80,000) but from distinct sources (human gastric mucosa, human erythrocyte membranes and rat spleen) were shown to have immunological cross-reactivity and comparable mobilities when subjected to polyacrylamide-gel electrophoresis under non-denaturing conditions. Kinetic parameters (kcat, Km and Ki) were determined for the interactions of the three enzymes with two synthetic chromogenic substrates and five inhibitors (naturally occurring and synthetic). On this basis it would appear that all of the enzymes should be considered equivalent to cathepsin E. pH-activity measurements indicated that the aspartic proteinase that originated from the erythrocyte membranes retained activity at a higher pH value than either of its readily soluble counterparts.

Derivatives of the 1-12 sequence of pig pepsinogen were prepared by solid-phase peptide synthesis. The three derivatives contain substitutions in the 4-7 region of the 1-12 sequence. Glycine was used to replace the hydrophobic residues -Val-Pro-Leu-Val- in pairs. After cleavage and purification, the synthetic peptides were compared with a synthetic peptide of the native sequence, prepared at the same time, with respect to their ability to inhibit the pepsin-catalysed clotting of milk. Inhibitory potency, determined from plots of percentage inhibition versus concentration of synthetic peptide, is inversely correlated with the substitution of glycine residues for the hydrophobic residues. Therefore the equilibrium inhibition of pepsin by these peptides is dominated by the hydrophobic nature of the 4-7 sequence region.