Crystalline Soybean Trypsin Inhibitor

Abstract

In a recent publication (1) the writer reported the isolation from soybean meal of a crystalline protein of the globulin type which inhibits the proteolytic action of trypsin. The method of isolation of the inhibitor crystals consists essentially in the following operations: Commercial soybean meal, defatted by a solvent extraction method by the manufacturer, is washed in the laboratory with 80 per cent ethyl alcohol. This washing facilitates the filtrations throughout the subsequent operations. The inhibitor, which is stable in acid, is then extracted from the meal with 0.25 N sulfuric acid. The acid extract is treated first with a small amount of bentonite which adsorbs some of the inert protein and is rejected, and then with a larger amount of bentonite-enough to adsorb all the inhibitor protein, leaving behind other materials in the acid extract. The inhibitor is eluted from the bentonite by means of a dilute aqueous pyridine solution, and the pyridine is removed by dialysis. The dialyzed solution is partly purified by adjusting the pH to 5.3 and filtering off the precipitate of inert material formed. The inhibitor protein is then precipitated from solution by adjusting the pH to 4.65 at 5-10°C. Finally a concentrated solution of the amorphous material is adjusted with acid to pH 5.1 and left at 36 °. Crystals in the form of fine needles and thin hexagonal plates gradually appear (Fig. 1). The crystallization is generally complete in 5 to 6 hours. The inhibitor crystals can be recrystallized from solution by addition of alcohol (Fig. 2). The crystals are then filtered, washed with cold acetone, and dried in the air at room temperature. The dried material refains its crystalline structure, is doubly refractive, and has the same inhibiting power on trypsin as the non-dried crystals isolated in the absence of alcohol. The details of the method of isolation are as follows :-1. Washing with 80 Per Cent Alcohol.-lO00 gm. cold-processed, defatted soybean meal I is added to a mixtule of 2400 ml. 95 per cent alcohol cooled to

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