Isoelectric Point Differences in Commercial Soybean Trypsin Inhibitors!

Isoelectric points of Kunitz' crystalline soybean trypsin inhibitor (STl), Bowman-Birk STI, and STI prepared by chromatography on diethylaminoethyI (DEAE)-cellulose were investigated by isoelectric focusing, gel filtration, ion-exchange resin treatment, solubility test, and titration. Results suggest that the STI of Kunitz may have an isoelectric point below pH 4.5. The isoelectric focusing pattern of the Kunitz inhibitor showed three peaks: at pH 3.5, 3.7, and 4.4. All three proteins were active inhibitors. Chromatography on anion-exchange resins separated the Kunitz STI into two fractions: one that eluted with wa ter and focused at pH 3.5 and another that eluted with increasing concentrations of NaCI solution and focused at pH 4.0. When ion-retardation resins were used only one peak resulted, focusing at pH 3.8. Solubility tests on the Kunitz preparation before and after treatment with anion-exchange resins supported evidence for different isoelectric points. Titration of Kunitz' STI at three levels of ionic strength gave an isoionic zone lower than pH 4.5. The Kunitz inhibitor contained about 20% impurities based on gel filtration and the purified protein focused at a single isoelectric pH of 4.0. The Bowman-Birk inhibitor consistently showed an isoelectric point of pH 4.2 in focusing and solubility tests, and it had little or no impurities on gel filtration. Although STI prepared by chromatography on DEAE-cellulose contained impurities, heterogeneity of isoelectric point (pH 4.0) was less than for Kunitz' STI. The multiple isoelectric points of crystalline STI were believed caused by interaction between protein and other constituents, possibly contaminants. Soybean trypsin inhibitor (STI) has been studied extensively. Since Kunitz (1) initially isolated and purified the inhibitor from soybeans, various chromatographic procedures have been applied to improve the isolation (2-6). At present, several purified proteins from soybeans, available commercially, are known to inhibit trypsin activity (7), although some of these preparations are believed to be impure (4,8). In 1969, Steiner and Frattali (7) summarized the purification and properties ofSTI. Kunitz reported the inhibitor as a globulin with a distinct minimum solubility at its isoelectric point of pH 4.5 (9). The isoelectric pH was established by solubility test and by cataphoretic mobility. Birk et al. (3) recorded pH 4.2 as the isoelectric point of the Bowman-Birk inhibitor as determined by solubility. Apparently these inhibitors are two different proteins with different isoelectric points (7). In an isoelectric focusing pattern, the Kunitz STI showed at least three protein peaks (10). Furthermore, during focusing this crystalline preparation yielded white precipitates, often observed in handling whey proteins. Attempts to remove these white precipitates led to a shift in isoelectric focusing pattern. Solubility tests on 1presented at the meeting of the American Society of Plant Physiologists, Bloomington, Ind., August 23 to 28, 1970. Contribution from the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. Copyright © 1971 American Association of Cereal Chemists, Inc., 1821 University Avenue, St. Paul, Minnesota 55104. All rights reserved.