The isolation of a crystalline trypsin inhibitor from soybean has been reported in previous publications (1). This paper deals with some of the properties of the new crystalline protein and with the mechanism of its inhibiting action on trypsin and chymotrypsin.The soybean inhibitor is a protein of the globulin type. It is precipitated by trichloracetic acid and is non-diffusable through collodion or cellophane membranes. Its light absorption spectrum is that of a typical protein with a maximum at 280 m/~ and a minimum at 252 m#. The protein contains less than 0.01 per cent phosphorus and is free of carbohydrate. It acts as an inhibitor only when it is in its native state; denaturation of the soy protein by heat, acid, or alkali is accompanied by a loss in its inhibiting power.The action of the native soybean protein as a trypsin inhibitor is due to its combination with trypsin to form an irreversible stoichiometric compound. The combination is apparently instantaneous.The soy protein inhibits slightly the proteolytic action of chymotrypsin, but unlike that of trypsin the inhibition is due to the formation of a loose reversible compound of the type described by Northrop (2) for the combination between pepsin or trypsin with crude inhibitors. The reaction between chymotrypsin and the soybean inhibitor was found to agree with the law of mass action, for a reversible uni-unimolecular reaction.Crystalline soybean protein, if denatured, is readily digestible by pepsin, by chymotrypsin, or by trypsin.Crystalline soybean inhibitor has no inhibiting effect either on the proteolytic activity or on the milk-clotting ability of pepsin. EXPEI~ rbtENTAL Test of Purity of Crystalline Soybean Trypsin Inhibitor 11. Effect of RecrystaUization.--The principal steps in the procedure for the x For the sake of brevity the terms "soy inhibitor" and "soy protein" are frequently used in the text instead of the full expression "crystalline soybean trypsin inhibitor."

A crystalline enzyme capable of digesting thymus nucleic acid (desoxyribonucleic acid) has been isolated from fresh beef pancreas. The enzyme called "desoxyribonuclease" is a protein of the albumin type. Its molecular weight is about 60,000 and its isoelectric point is near pH 5.0. It contains about 8 per cent tyrosine and 2 per cent tryptophane. It is readily denatured by heat. The denaturation is reversible if heated in dilute acid at pH about 3.0. The digestion of thymus nucleic acid by crystalline desoxyribonuclease is accompanied by a gradual increase in the specific absorption of ultraviolet light by the acid. The spectrophotometric measurement of the rate of increase in the light absorption can be conveniently used as a general method for estimating desoxyribonuclease activity. Details are given of the method for isolation of crystalline desoxyribonuclease and of the spectrophotometric procedure for the measurement of desoxyribonuclease activity.

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

The isolation of trypsin from active pancreatic extract (1) and of chymo-trypsinogen and chymo-trypsin (2) 1 from fresh inactive pancreas has been described in previous papers. Crystalline trypsinogen has also been obtained (3) from inactive cattle pancreas and transformed into active trypsin which may then be crystallized much more readily than by the earlier method. During the course of this work a polypeptide, which has a powerful inhibiting effect on trypsin, as well as a compound of this substance with trypsin, was also obtained in crystalline form (4). The present paper contains detailed descriptions of methods of preparing these substances and a brief description of their properties. G E N E R A L P~ROPERTIES TrypsinogenTrypsinogen is obtained as small triangular prisms (Fig. 1). When these crystals are dissolved in neutral solution the trypsinogen is rapidly transformed into active trypsin and it has, therefore, been impossible so far to recrystallize trypsinogen. The original crystallization occurs without activation owing to the presence of the inhibitor and if inhibitor is added to a solution of trypsinogen recrystallization may be carried out without activation. Numerous attempts have been made to recrystallize inhibitor-free trypsinogen under conditions 1 It has recently been found that poorly formed needle-shaped protein crystals, which have about the same activity as the usual chymo-trypsin crystals, may be obtained from the mother liquor of the chymo-trypsin crystallization. The properties of these new crystals are now being investigated.

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