Studies on gastric proteolysis. 2. The nature of the enzyme–substrate interaction responsible for gastric proteolytic pH–activity curves with two maxima

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Abstract
Fractionation of human gastric juice and swine gastric-mucosal extracts with ammonium sulfate, sodium chloride and magnesium sulfate has failed to achieve separation of enzymes responsible for the 2 proteolytic pH maxima which these materials exert at values below pH 5.0; nor could separation be achieved by adsorption on calcium phosphate gel, on ion-exchange resins, by electrophoresis in the Tiselius apparatus and by differential ultra -centrifuging. During electrophoresis of gastric juice, 2 anodal-moving homogeneous components at pH 2.5 were isolated. Each digested proteins with two pH maxima below pH 5.0. The maxima of the smaller component occurred at lower pH values than the corresponding maxima of the larger. Hydrogen sulfide caused an increased liberation of amino groups by gastric juice from human serum albumin at all values between pH 1.5 and 5.0. Cysteine was without effect on proteolysis over this range. The ratio of activity at the 2 pH maxima was changed during treatment of gastric juice with salt solutions, indicating that one maximum could be altered independently of the other. Paper-chromatographic analysis revealed that the end products of digestion at the 2 maximal pH values are different. Crystalline swine pepsin, like human gastric juice, digests plasma protein, bovine crystalline plasma albumin and bovine plasma albumin fraction V, with two pH maxima below pH 5.0, but egg albumin with only one. Pepsin A was isolated from crystalline pepsin, was found to be homogeneous by paper-electrophoretic and solubility tests, and to digest single component substrates with two maxima. Attempts to explain the existence of 2 pH maxima in terms of enzyme-substrate union at a single site were unsuccessful. It is suggested that the 2 proteolytic pH maxima which may be exhibited by gastric juice and gastric mucosal extracts below pH 5.0 result from the action of a single enzyme, pepsin, which achieves this effect because it possesses two sorts of active centre, each of which attacks maximally at a different pH a different type of substrate site.