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1.
A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.2 per day, corresponding to a molecular radius of 2.6 x 10–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8. 相似文献
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John H. Northrop 《The Journal of general physiology》1932,16(2):339-348
The rate of digestion, as determined by the increase in non-protein nitrogen or formol titration, of casein, gelatin, and hemoglobin with crystalline trypsin preparations increases nearly in proportion to the concentration of protein, but with crude pancreatic extract the rate of digestion becomes independent of the protein concentration in concentrations of more than 2.5 per cent. With both enzymes the rate of digestion of mixtures of 5 per cent casein and gelatin is greater than would be expected from the point of view of a compound between enzyme and substrate. The rate of digestion of 5 per cent casein in the presence of 5 per cent gelatin is exactly the same as that of 5 per cent casein alone. This result is obtained with both enzymes. The digestion of casein with crude trypsin follows the course of a monomolecular reaction quite closely while with purified trypsin the velocity constant decreases as the reaction proceeds. In the case of hemoglobin the monomolecular velocity constant decreases with both purified and crude enzyme. When the reaction is followed by changes in the viscosity of the solution the abnormal effect of changing substrate concentration disappears and the reaction is in fair agreement with the monomolecular equation. The results as a whole indicate that the abnormalities of the reaction are due to the occurrence of several consecutive reactions rather than to the formation of a substrate enzyme compound. 相似文献
4.
Methods are described for the isolation and crystallization of trypsinogen, trypsin, a substance which inhibits trypsin, and an inhibitor-trypsin compound. Analyses and some of the properties of these compounds are given. 相似文献
5.
M. Kunitz 《The Journal of general physiology》1947,30(4):311-320
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. 相似文献
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1. The rate of inactivation of crystalline trypsin solutions and the nature of the products formed during the inactivation at various pH at temperatures below 37°C. have been studied. 2. The inactivation may be reversible or irreversible. Reversible inactivation is accompanied by the formation of reversibly denatured protein. This denatured protein exists in equilibrium with the native active protein and the equilibrium is shifted towards the denatured form by raising the temperature or by increasing the alkalinity. The decrease in the fraction of active enzyme present (due to the formation of this reversibly denatured protein) as the pH is increased from 8.0 to 12.0 accounts for the decrease in the rate of digestion of proteins by trypsin in this range of pH. 3. The loss of activity at high temperatures or in alkaline solutions, just described, is very rapid and is completely reversible for a short time only. If the solutions are allowed to stand the loss in activity becomes gradually irreversible and is accompanied by the appearance of various reaction products the nature of which depends upon the temperature and pH of the solution. 4. On the acid side of pH 2.0 the trypsin protein is changed to an inactive form which is irreversibly denatured by heat. The course of the reaction in this range is monomolecular and its velocity increases as the acidity increases. 5. From pH 2.0 to 9.0 trypsin protein is slowly hydrolyzed. The course of the inactivation in this range of pH is bimolecular and its velocity increases as the alkalinity increases to pH 10.0 and then decreases. As a result of these two reactions there is a point of maximum stability at about pH 2.3. 6. On the alkaline side of pH 13.0 the reaction is similar to that in strong acid solution and consists in the formation of inactive protein. The course of the reaction is monomolecular and the velocity increases with increasing alkalinity. From pH 9.0 to 12.0 some hydrolysis takes place and some inactive protein is formed and the course of the reaction is represented by the sum of a bi- and monomolecular reaction. The rate of hydrolysis decreases as the solution becomes more alkaline than pH 10.0 while the rate of formation of inactive protein increases so that there is a second point at about pH 13.0 at which the rate of inactivation is a minimum. In general the decrease in activity under all these conditions is proportional to the decrease in the concentration of the trypsin protein. Equations have been derived which agree quantitatively with the various inactivation experiments. 相似文献
7.
John H. Northrop 《The Journal of general physiology》1932,16(2):323-337
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. 相似文献
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大豆胰蛋白酶抑制剂对棉铃虫幼虫消化生理和生长发育的影响 总被引:21,自引:2,他引:21
本研究根据棉铃虫Helicotverpa ormigera(Hubner)幼虫中肠蛋白酶在离体条件下对蛋白酶抑制剂的反应,选择具有较强抑制作用的大豆胰蛋白酶抑制剂,以0.21-4.2%(干重)的浓度配入幼虫人工饲料,测定了幼虫短期和长期取食这些饲料引起的中肠类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力的变化和生长抑制效应。短期取食抑制剂的幼虫,中肠弱碱性类胰蛋白酶活力显著增高,在4.2%。浓度下比对照高出21%;强碱性类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力显著降低,生长发育受到明显抑制。长期取食低浓度(0.84%)抑制剂的幼虫,弱碱性类胰蛋白酶和类胰凝乳蛋白酶活力显著增高,强碱性类胰蛋白酶活力显著降低。总蛋白酶活力变化不显著;长期取食高浓度(4.2%)抑制剂的幼虫,强碱性类胰蛋白酶和总蛋白酶活力显著降低,其它酶活力变化不显著。抑制剂随浓度的增高对幼虫生长的抑制作用加强,但浓度高于0.84%后,抑制强度的变化减小。据此作者认为,蛋白酶抑制剂对昆虫抗营养效应在于它对蛋白酶的激活和抑制作用,从而导致各种蛋白酶间的协调性破坏,昆虫消化过程受阻,影响生长发育。 相似文献
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Henry W. Scherp 《The Journal of general physiology》1933,16(5):795-800
The diffusion coefficient of crystalline trypsin in 0.5 saturated magnesium sulfate at 5°C. is 0.020 ±0.001 cm.2 per day, corresponding to a molecular radius of 2.6 x 10–7 cm. The rate of diffusion of the proteolytic activity is the same as that of the protein nitrogen, indicating that these two properties are held together in chemical combination and not in the form of an adsorption complex. 相似文献
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大豆胰蛋白酶抑制剂与棉酚或丹宁混用对棉铃虫中肠蛋白酶和生长率的影响 总被引:4,自引:0,他引:4
本文报告了大豆胰蛋白酶抑制剂(STl)与棉酚、丹宁酸单一和协同作用对棉铃虫Helicoverpa armigera(Hubner)幼虫中肠蛋白酶活性和生长速率的影响。在离体条件下,STI、棉酚和丹宁酸均对中肠蛋白酶有抑制作用,以STI的作用最强。活体试验表明,人工饲料中0.84%(干重)的S丁I对强碱性类胰蛋白酶活力有显著抑制作用;0.3%丹宁酸则对弱碱性类胰蛋白酶和总蛋白酶活力有显著抑制作用;0.3%棉酚对几种蛋白酶活力的影响均不显著。三者均能显著抑制幼虫的生长,而Sn与棉酚或丹宁酸的协同作用比三者的单独作用更能有效地抑制幼虫的生长发育和中肠蛋白酶活性。 相似文献
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大豆胰蛋白酶抑制剂与棉酚或丹宁混用对棉铃虫中肠蛋白酶和生长率的影响 总被引:4,自引:0,他引:4
本文报告了大豆胰蛋白酶抑制剂(STl)与棉酚、丹宁酸单一和协同作用对棉铃虫Helicoverpa armigera(Hubner)幼虫中肠蛋白酶活性和生长速率的影响。在离体条件下,STI、棉酚和丹宁酸均对中肠蛋白酶有抑制作用,以STI的作用最强。活体试验表明,人工饲料中0.84%(干重)的S丁I对强碱性类胰蛋白酶活力有显著抑制作用;0.3%丹宁酸则对弱碱性类胰蛋白酶和总蛋白酶活力有显著抑制作用;0.3%棉酚对几种蛋白酶活力的影响均不显著。三者均能显著抑制幼虫的生长,而Sn与棉酚或丹宁酸的协同作用比三者的单独作用更能有效地抑制幼虫的生长发育和中肠蛋白酶活性。 相似文献
14.
David Grob 《The Journal of general physiology》1943,26(4):423-430
1. Serum antitrypsin and pancreatic trypsin inhibitor inhibited the coagulation of plasma in vitro. 2. This could be largely prevented by trypsin. 3. The anticoagulant action of the trypsin inhibitor was apparently due to its antiprothrombic action. It had no appreciable antithrombic action. 4. Examination of the blood of two hemophiliacs indicated that the prolonged coagulation time of their blood is not due to an excess of trypsin inhibitor. 5. Examination of the blood of heparinized dogs indicated that heparin does not appreciably contribute to the antiproteolytic activity of the serum. 相似文献
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海芋胰蛋白酶抑制剂的分离纯化及性质研究 总被引:3,自引:1,他引:3
利用亲和层析和分子筛凝胶过滤等技术,从海芋根茎中分离纯化到一种胰蛋白酶抑制剂,简称AMTI。经PAGE、SDS-PAGE和Western blot鉴定均显示单一条带,经SDS-PAGE测定,其分子量为22000,经等电聚焦(IEF)测定,其等电点为6.2。根据对胰蛋白酶的抑制比可知该抑制剂为单头抑制剂,其抑制活性在60℃和pH5 ̄11范围内保持稳定。 相似文献
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M. Kunitz 《The Journal of general physiology》1939,22(4):429-446
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. 相似文献
17.
A new crystalline protein, chymo-trypsinogen, has been isolated from acid extracts of fresh cattle pancreas. This protein is not an enzyme but is transformed by minute amounts of trypsin into an active proteolytic enzyme called chymo-trypsin. The chymo-trypsin has also been obtained in crystalline form. The chymo-trypsinogen cannot be activated by enterokinase, pepsin, inactive trypsin, or calcium chloride. There is an extremely slow spontaneous activation upon standing in solution. The activation of chymo-trypsinogen by trypsin follows the course of a monomolecular reaction the velocity constant of which is proportional to the trypsin concentration and independent of the chymotrypsinogen concentration. The rate of activation is a maximum at pH 7.0–8.0. Activation is accompanied by an increase of six primary amino groups per mole but no split products could be found, indicating that the activation consists in an intramolecular rearrangement. There is a slight change in optical activity but no change in molecular weight. The physical and chemical properties of both proteins are constant through a series of fractional crystallizations. The activity of chymo-trypsin decreases in proportion to the destruction of the native protein by pepsin digestion or denaturation by heat or acid. Chymo-trypsin has powerful milk-clotting power but does not clot blood plasma and differs qualitatively in this respect from the crystalline trypsin previously reported. It hydrolyzes sturin, casein, gelatin, and hemoglobin more slowly than does crystalline trypsin but the hydrolysis of casein is carried much further. The hydrolysis takes place at different linkages from those attacked by trypsin. The optimum pH for the digestion of casein is about 8.0–9.0. It does not hydrolyze any of a series of dipeptides or polypeptides tested. Several chemical and physical properties of both proteins have been determined. 相似文献
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The Physico-chemical properties of an algal cytochrome, Porphyratenera cytochrome 553 were investigated, using a crystallinepreparation of the substance. The cytochrome was found to behighly resistant to heat and preservation. The stability ofthe cytochrome was also prominent in a wide range of pH. Absorption spectra as: well as data concerning the extinctioncoefficients of the oxidized and reduced forms are presented. The oxidation-reduction potential of the cytochrome was determinedat various pH. The value for the normal potential was foundto decrease gradually with increasing pH; from 355 mV at pH5.0, to 270 mV at pH 11.0. Electrophoretic investigation revealedthat this cytochrome is a markedly acidic protein, with an isoelectricpoint of pH 3.5. The molecular weight of the cytochrome wasdetermined from ultracentrifugal and diffusion experiments tobe 13,600. The values for the minimum molecular weight calculatedfrom the heme- and iron-contents are, respectively, 12,000 and11,000. (Received April 20, 1960; ) 相似文献
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John H. Northrop 《The Journal of general physiology》1922,4(3):245-260
1. A study has been made of the equilibrium existing between trypsin and the substances formed in the digestion of proteins which inhibit its action. 2. This substance could not be obtained by the hydrolysis of the proteins by acid or alkali. It is dialyzable. 3. The equilibrium between this substance (inhibitor) and trypsin is found to agree with the equation, trypsin + inhibitor ⇌ trypsin-inhibitor The equilibrium is reached instantaneously and is independent of the substrate concentration. If it be further assumed that the rate of hydrolysis is proportional to the concentration of the free trypsin and that the equilibrium conforms to the law of mass action, it is possible to calculate the experimental results by the application of the law of mass action. 4. The equilibrium has been studied by varying (a) the concentration of the inhibiting substance, (b) the concentration of trypsin, (c) the concentration of gelatin, and (d) the concentration of trypsin and inhibitor (the relative concentration of the two remaining the same). In all cases the results agree quantitatively with those predicted by the law of mass action. 5. It was found that the percentage retarding effect of the inhibiting substance on the rate of hydrolysis is independent of the hydrogen ion concentration between pH 6.3 and 10.0. 6. The fact that the experimental results agree with the mechanism outlined under 3, is contrary to the assumption that any appreciable amount of trypsin is combined with the gelatin at any one time; i.e., the velocity of the hydrolysis must depend on the time required for such a compound to form rather than for it to decompose. 7. The experiments may be considered as experimental proof of the validity of Arrhenius'' explanation of Schütz''s rule as applied to trypsin digestion. 8. Inactivated trypsin does not enter into the equilibrium. 相似文献
20.
Sam Granick 《The Journal of general physiology》1942,25(4):571-578
1. Guinea pig hemoglobin crystals are shown to be readily permeable to ferricyanide and hydrosulfite, indicating the presence of interstices between the protein molecules of the crystal. 2. The assumptions of closest hexagonal packing and of spherical molecules of HbO2 lead to a crystal lattice having interstices between the molecules which represent 25 per cent by volume of the crystal. These spaces would be just large enough at their narrowest junctures to permit ferricyanide ions to diffuse through them. If these spaces were filled with water, then 20 per cent by weight of the crystals would be water. 3. The hemes are on the surface of the globin and are arranged facing the interstices between the molecules of the lattice. 4. The binding of O2 in HbO2 is stronger in the crystal lattice than in solution. 5. Hydrogen, activated with colloidal palladium, will not reduce ferrihemoglobin except in the presence of a redox dye. 6. In the reduction of O2 by activated hydrogen, H2O2 can be demonstrated by the formation of the H2O2-ferrihemoglobin spectrum. 相似文献