CRYSTALLINE PEPSIN : VI. INACTIVATION BY BETA AND GAMMA RAYS FROM RADIUM AND BY ULTRA-VIOLET LIGHT

1. The loss in activity of crystalline pepsin solutions when exposed to beta and gamma rays from radium or to ultra-violet light is accompanied by a corresponding decrease in pepsin protein. 2. The rate of inactivation by ultra-violet light depends upon the pH and is a maximum at about pH 2.0.     

THE TEMPERATURE COEFFICIENT OF INACTIVATION OF CRYSTALLINE PEPSIN BY ULTRA-VIOLET RADIATION

Determinations of the temperature coefficient of inactivation of pure crystalline pepsin solutions by ultra-violet irradiation give values very close to unity (1.02).     

COMPARATIVE HYDROLYSIS OF GELATIN BY PEPSIN,TRYPSIN, ACID,AND ALKALI

A comparison has been made of the relative velocity of hydrolysis of the various peptid linkings of the gelatin molecule when hydrolyzed by acid, alkali, pepsin or trypsin. It has been found that: 1. Those linkages which are most rapidly split by pepsin or trypsin are among the more resistant to acid hydrolysis. 2. Those linkages which are hydrolyzed by pepsin are also hydrolyzed by trypsin. 3. Trypsin hydrolyzes linkages which are not attacked by pepsin. 4. Of the linkages which are hydrolyzed by both enzymes, those which are most rapidly hydrolyzed by pepsin are only slowly attacked by trypsin. 5. Those linkages which are attacked by trypsin or pepsin are among the ones first (most rapidly) hydrolyzed by alkali. In general it may be said that the course of the early stages of hydrolysis of gelatin is similar with alkali, trypsin, or pepsin and quite different with acid.     

CRYSTALLINE TRYPSIN : IV. REVERSIBILITY OF THE INACTIVATION AND DENATURATION OF TRYPSIN BY HEAT

1. If dilute solutions of purified trypsin of low salt concentration at pH from 1 to 7 are heated to 100°C. for 1 to 5 minutes and then cooled to 20°C. there is no loss of activity or formation of denatured protein. If the hot trypsin solution is added directly to cold salt solution, on the other hand, all the protein precipitates and the supernatant solution is inactive. 2. The per cent of the total protein and activity present in the soluble form decreases from 100 per cent to zero as the temperature is raised from 20°C. to 60°C. and increases again from zero to 100 per cent as the solution is cooled from 60°C. to 20°C. The per cent of the total protein present in the soluble (native) form at any one temperature is nearly the same whether the temperature is reached from above or below. 3. If trypsin solutions at pH 7 are heated for increasing lengths of time at various temperatures and analyzed for total activity and total protein nitrogen after cooling, and for soluble activity and soluble (native) protein nitrogen, it is found that the soluble activity and soluble protein nitrogen decrease more and more rapidly as the temperature is raised, in agreement with the usual effects of temperature on the denaturation of protein. The total protein and total activity, on the other hand, decrease more and more rapidly up to about 70°C. but as the temperature is raised above this there is less rapid change in the total protein or total activity and at 92°C. the solutions are much more stable than at 42°C. 4. Casein and peptone are not digested by trypsin at 100°C. but when this digestion mixture is cooled to 35°C. rapid digestion occurs. A solution of trypsin at 100°C. added to peptone solution at zero degree digests the peptone much less rapidly than it does if the trypsin solution is allowed to cool slowly before adding it to the peptone solution. 5. The precipitate of insoluble protein obtained from adding hot trypsin solutions to cold salt solutions contains the S-S groups in free form as is usual for denatured protein. 6. The results show that there is an equilibrium between native and denatured trypsin protein the extent of which is determined by the temperature. Above 60°C. the protein is in the denatured and inactive form and below 20°C. it is in the native and active form. The equilibrium is attained rapidly. The results also show that the formation of denatured protein is proportional to the loss in activity and that the re-formation of native protein is proportional to the recovery of activity of the enzyme. This is strong evidence for the conclusion that the proteolytic activity of the preparation is a property of the native protein molecule.     

CRYSTALLINE PEPSIN : III. PREPARATION OF ACTIVE CRYSTALLINE PEPSIN FROM INACTIVE DENATURED PEPSIN

1. Pepsin solutions which have been completely denatured and inactivated by adjusting to pH 10.5 recover some of their activity when titrated to about pH 5.4 and allowed to stand at 22°C. for 24 to 48 hours. 2. Control experiments show that this inactivation and reactivation are probably not due to the effect of any inhibiting substance. 3. A method of isolation of the reactivated material has been worked out. 4. The reactivated material recovered in this way is a protein with the same general solubility, the same crystalline form, and the same specific proteolytic activity as the original crystalline pepsin. 5. This furnishes additional proof that the proteolytic activity is a property of the protein molecule.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity.     

INACTIVATION OF PEPSIN BY IODINE : II. ISOLATION OF CRYSTALLINEl-MONO-IODOTYROSINE FROM PARTIALLY IODINATED PEPSIN

1. Pepsin solutions were iodinated at pH 5.0–6.0 until 10–20 per cent of the activity was lost and 1/20 (0.7 per cent) of the saturating amount of iodine had been introduced into the protein molecule. After alkaline hydrolysis 65 per cent of the original iodine was accounted for as mono-iodotyrosine although only 42 per cent was isolated as a crystalline product. No evidence was obtained to support the possibility that any group other than tyrosine in pepsin was iodinated. 2. Some of the properties of the crystalline l-mono-iodotyrosine were determined and compared to those of di-iodotyrosine. 3. One iodinated pepsin preparation was crystallized. The crystal form was the same as that of the original pepsin. A solubility curve of the crystals demonstrated that it was very different from pepsin and had nearly constant solubility.     

CRYSTALLINE PEPSIN : V. ISOLATION OF CRYSTALLINE PEPSIN FROM BOVINE GASTRIC JUICE

1. A method has been described for isolating a crystalline protein with high proteolytic activity from bovine gastric juice by means of precipitation with magnesium sulfate and fractionation of the precipitate with acetone and magnesium sulfate. 2. The crystalline protein obtained in this way has the same crystalline form, optical activity, and specific activity, as determined by a number of methods, as does the crystalline protein previously isolated from swine gastric mucosa. 3. The solubility of the two preparations, however, is additive so that they are different although very closely related proteins.     

THE ABSORPTION OF ULTRA-VIOLET RADIATION BY CRYSTALLINE PEPSIN

Determination of the absorption spectra of pure preparations of Northrop''s crystalline pepsin inactivated by irradiation with ultra-violet light shows that the total absorption in the ultra-violet region of the spectrum increases with the degree of inactivation. This increase is especially marked between 2400 and 2750 Å.u. The rate of photoinactivation is shown to be sensitive to changes in pH, increasing with lower values, and evidently bears a one-quantum relationship to the energy flux. Tests of the rate of inactivation of pepsin exposed to several different bands of the ultra-violet spectrum, in relation to the absorbed energy, indicate that the destruction spectrum of the enzyme agrees essentially with its absorption spectrum and is similar to that of urease.     

THE REVERSIBLE INACTIVATION OF TOBACCO MOSAIC VIRUS BY CRYSTALLINE RIBONUCLEASE

The reversible inactivation of tobacco mosaic virus by crystalline ribonuclease is reported. Studies on the effect of time of standing on the amount of inactivation, and on the effect of dilution and repeated high speed centrifugation on the recovery of virus activity, and the preparation of an insoluble virus-enzyme complex show that the inactivation is brought about at least in part by a combination between virus and enzyme. The significance of the fact that ribonuclease has no detectable effect on the virus nucleic acid when the latter is in combination with protein in the form of virus is discussed with respect to the structure of the virus.     

FRACTIONATION OF PEPSIN : I. ISOLATION OF CRYSTALLINE PEPSIN OF CONSTANT ACTIVITY AND SOLUBILITY FROM PEPSINOGEN OR COMMERCIAL PEPSIN PREPARATIONS. II. PREPARATION OF A LESS SOLUBLE FRACTION. III. SOLUBILITY CURVES OF MIXTURES OF THE SOLUBLE AND INSOLUBLE FRACTIONS. IV. PREPARATION OF HIGHLY ACTIVE PEPSIN FROM PEPSINOGEN

1. Solubility curves of crude pepsin preparations indicate the presence of more than one protein. 2. One of these proteins has been isolated and crystallized and found to have constant activity and constant solubility in several solvents. 3. The solubility measurements are complicated by the unstable nature of the protein and the fact that in certain solvents the solubility of the protein is markedly affected by the presence of non-protein nitrogen decomposition products while in others this is not the case. 4. A more insoluble protein has been prepared of lower solubility and lower activity, as measured by hemoglobin digestion. The activity, as measured by the digestion of other proteins, is about the same as the more soluble fraction. This insoluble fraction does not have constant solubility. 5. Mixtures of the insoluble and the soluble fractions give preparations having rounded solubility curves typical of solid solutions and resembling very closely those of the original preparation. 6. A small amount of pepsinogen and pepsin from pepsinogen has been separated which has nearly twice the enzymatic activity on hemoglobin as does the most active pepsin previously isolated.     

THE INACTIVATION OF TRYPSIN : IV. THE ADSORPTION OF TRYPSIN BY CHARCOAL.

1. Charcoal removes trypsin from solution. The amount removed depends on the order in which the solutions are mixed. The reaction is not reversible and is almost independent of the pH of the solution. 2. Charcoal which has been previously treated with gelatin does not remove trypsin from solution. 3. The reaction is not analogous either to the reaction between trypsin and the inhibiting substance of serum or to the reaction between solid protein and either pepsin or trypsin.     

THE INFLUENCE OF THE SUBSTRATE CONCENTRATION ON THE RATE OF HYDROLYSIS OF PROTEINS BY PEPSIN

1. It is pointed out that the apparent exceptions to the law of mass action found in enzyme reactions may be found in catalytic reactions in strictly homogeneous solutions. 2. These deviations in the rate of reaction from the law of mass action may be explained by the hypothesis that the active mass of the reacting substances is not directly proportional to the total concentration of substance taken. 3. In support of this suggestion it is shown that for any given concentration of pepsin the relative rate of digestion of concentrated and of dilute protein solutions is always the same. If the rate of digestion depended on the saturation of the surface of the enzyme by substrate the relative rate of digestion of concentrated protein solutions should increase more rapidly with the concentration of enzyme than that of dilute solutions. This was found not to be true, even when the enzyme could not be considered saturated in the dilute protein solutions. 4. The rate of digestion and the conductivity of egg albumin solutions of different concentration were found to be approximately proportional at the same pH. This agrees with the hypothesis first expressed by Pauli that the ionized protein is largely or entirely the form which is attacked by the enzyme. 5. The rate of digestion is diminished by a very large increase in the viscosity of the protein solution. This effect is probably a mechanical one due to the retardation of the diffusion of the enzyme.     

THE PHOSPHATE ION AND HYDROLYSIS BY PANCREATIC LIPASE

The equation, (activity of enzyme) (pPO₄)ⁿ = K, has been investigated and has been shown to have only a limited application to the effect of the phosphate ion on the hydrolytic activity of pancreatic lipase. The deviations observed are ascribed to the effect of certain factors on the stability of the enzyme.     

THE EFFECT OF RADIOACTIVE RADIATIONS AND X-RAYS ON ENZYMES : VI. THE INFLUENCE OF VARIATION OF TEMPERATURE UPON THE RATE OF RADIOCHEMICAL INACTIVATION OF SOLUTIONS OF PEPSIN BY BETA RADIATION.

Data are presented which indicate that variation in temperature is associated with only slight variation in the speed of the radiochemical inactivation of pepsin in dilute solution.     

酸催化半干微波法水解蚕蛹蛋白的研究

以酸为催化剂,采用半干微波法水解蚕蛹蛋白制备氨基酸。在适宜条件下,20min内氨基氮生成率达52.4％,产品收率为43.7％,游离氨基酸收率为34.0％。与常规酸水解法相比,反应时间大大缩短,能耗大幅度下降,产品收率和纯度提高。