Action of Rennin and Pepsin on β-Casein: Insoluble and Soluble Products

Authors:
  • JCJ. Cerbulis
    J. Cerbulis
  • JCJ.H. Custer
    J.H. Custer
  • CZC.A. Zittle
    C.A. Zittle
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Abstract
B-Casein was hydrolyzed to a considerable extent by the enzymes pepsin and rennin at pH 6.4. Pepsin hydrolyzed B-casein more extensively than rennin when given sufficient time. Hydrolysis of B-casein was evidenced by an increase in solubility at pH 4.7, and an increase in solubility in 2% trichloroacetic acid (TCA). Solubility in 12% TCA was about the same before and after enzyme treatment; hence, it was not a suitable reagent for demonstrating hydrolysis. The insoluble portion (pH 4.7; 2% TCA) of ~-casein after enzyme treatment was heterogeneous, containing at least three components. The soluble fraction was very heterogeneous and contained ten or more components. The major products released by rennin and pepsin have similar electrophoretic behavior, but there were differences among the minor products. Reports on the action of the enzymes rennin and pepsin on B-casein have been diverse. Some have concluded that only a-casein is altered by rennin, whereas B-casein and ~,-casein are not attacked (3, 1), but are merely coprecipitated unchanged along with the altered a-casein in the presence of calcium salts (3). Others have reported that B-casein shows only a slow general proteolysis (6, 7); that the prolonged action of rennin on B-casein gives a product no longer preeipitable with calcium ion (9) ; and that B-casein is split into two components in 4 hr. by rennin (8). A recent report has shown that B-casein is solubilized considerably by pepsin in 5 rain. (10). The present investigation was undertaken to learn more about the action of the enzymes rennin and pepsin on B-casein, and to clarify the reports just cited. Since some of the conflict appeared to be due to the choice of conditions for precipitating the treated B-casein, precipitation at pH 4.7 as well as precipitation with two concentrations (2 and 12%) of trichloroacetic acid (TCA) were compared. The components of the insoluble and soluble fractions were investigated at the same time by eleetrophoresis and chromatography. EXPERIMENTAL PROCEDURE Enzymes. Pepsin was a crystalline, Commercial product. Rennin was a highly purified product obtained through the courtesy of Dr. R. A. Sullivan, National Dairy Research Laboratories, L. I., N. ¥. This rennin had the same specific activity as crystalline rennin. B-Casein was prepared by the method of Hipp et al. (5), utilizing differential solubility in aqueous urea solutions. Preparation of dry fractions. B-Casein and all precipitates were dried by
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