FORMATION OF NEW CRYSTALLINE ENZYMES FROM CHYMOTRYPSIN : ISOLATION OF BETA AND GAMMA CHYMOTRYPSIN

A solution of chymotrypsin on slight hydrolysis undergoes an irreversible change into new proteins, two of which are enzymes and have been isolated in crystalline form. The new crystalline enzymes, called beta and gamma chymotrypsins, differ from the original chymotrypsin as well as from each other in many physical and chemical respects, such as molecular weight, crystalline form, solubility, and combining capacity with acid. The new enzymes still possess the same enzymatic properties as chymotrypsin. It thus appears that the irreversible change from chymotrypsin to the new enzymes does not affect the structure responsible for the enzymatic activity of the molecule. The solubility curves of the new enzymes agree approximately with the curves for a solid phase of one component and furnish very good evidence that the preparations represent distinct substances. The various enzymes when mixed at the proper pH have a tendency to form mixed crystals of the solid solution type. Thus at pH 4.0 gamma chymotrypsin combines to form solid solution crystals with either alpha or beta chymotrypsin. Hence at this pH separation of gamma from either alpha or beta by means of fractional crystallization is impossible. At pH 5.0–6.0, however, each material crystallizes in its own characteristic form and at its own rate; thus a fractional separation of the various enzymes from each other becomes feasible

CRYSTALLIZATION OF SALT-FREE CHYMOTRYPSINOGEN AND CHYMOTRYPSIN FROM SOLUTION IN DILUTE ETHYL ALCOHOL

Chymotrypsinogen and chymotrypsin crystallize readily from dilute solutions of ethyl alcohol in the absence of salts. The crystals formed in the presence of alcohol differ in appearance from those formed in the presence of ammonium sulfate. Chymotrypsinogen yields well formed polyhedrons instead of fine needles usually produced in ammonium sulfate solution. Chymotrypsin yields fine needles in the presence of alcohol and rhombohedrons in the presence of ammonium sulfate. The enzymatic properties of the crystals formed in the presence of alcohol are identical with those of the crystals isolated in the presence of ammonium sulfate

ISOLATION OF A CRYSTALLINE PROTEIN COMPOUND OF TRYPSIN AND OF SOYBEAN TRYPSIN-INHIBITOR

A crystalline protein compound has been isolated from a solution containing crystalline trypsin and crystalline soybean inhibitor. The protein consists of about equal weights of trypsin and of the inhibitor. Denaturation by heat or by alkali resolves the compound into its components

FORMATION OF TRYPSIN FROM CRYSTALLINE TRYPSINOGEN BY MEANS OF ENTEROKINASE

Crystalline trypsinogen is most readily and completely transformed into trypsin by means of enterokinase in the range of pH 5.2–6.0 at 5°C. and at a concentration of trypsinogen of not more than 0.1 mg. per ml. The action of enterokinase under these conditions is that of a typical enzyme. The process follows closely the course of a catalytic unimolecular reaction, the rate of formation of trypsin being proportional to the concentration of enterokinase added and the ultimate amount of trypsin formed being independent of the concentration of enterokinase. The catalytic action of enterokinase on crystalline trypsinogen in dilute solution at pH more alkaline than 6.0 and in concentrated solution at pH even slightly below 6.0 is complicated by the partial transformation of the trypsinogen into inert protein which can no longer be changed into trypsin even by a large excess of enterokinase. This secondary reaction is catalyzed by the trypsin formed and the rate of the reaction is proportional to the concentration of trypsin as well as to the concentration of trypsinogen in solution. Hence under these conditions only a small part of the trypsinogen is changed by enterokinase into trypsin while a considerable part of the trypsinogen is transformed into inert protein, the more so the lower the concentration of enterokinase used. The kinetics of the formation of trypsin by means of enterokinase when accompanied by the formation of inert protein can be explained quantitatively on the theoretical assumption that both reactions are of the simple catalytic unimolecular type, the catalyst being enterokinase in the first reaction and trypsin in the second reaction

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van't Hoff's law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H2O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin

FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q5–15 = 1.70 to Q25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q10 = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000

CRYSTALLINE RIBONUCLEASE

1. A crystalline enzyme capable of digesting yeast nucleic acid has been isolated from fresh beef pancreas. 2. The enzyme called "ribonuclease" is a soluble protein of albumin type. Its molecular weight is about 15,000. Its isoelectric point is in the region of pH 8.0. 3. Ribonuclease splits yeast nucleic acid into fragments small enough to diffuse readily through collodion or cellophane membranes. 4. The split products of digestion, unlike the undigested yeast nucleic acid, are not precipitable with glacial acetic acid or dilute hydrochloric acid. 5. The digestion of yeast nucleic acid is accompanied by a gradual formation of free acid groups without any significant liberation of free phosphoric acid. 6. Ribonuclease is stable over a wide range of pH even when heated for a short time at 100°C. Its maximum stability is in the range of pH 2.0 to 4.5. 7. Denaturation of the protein of ribonuclease by heat or alkali, or digestion of the protein by pepsin, causes a corresponding percentage loss in the enzymatic activity of the material

CRYSTALLINE INORGANIC PYROPHOSPHATASE ISOLATED FROM BAKER'S YEAST

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40°C. and pH 7.0 at a concentration of 3 to 4 x 10–3 M Na4P2O7 and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na4P2O7 or K4P2O7 proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme