CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR : II. GENERAL PROPERTIES

A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ.     

A crystalline protein-proteinase inhibitor from pinto bean seeds.

A crystalline protein-proteinase inhibitor has been isolated from seeds of Pinto bean (Phaseolus vulgaris cultvar. Pinto). It has an average molecular weight of 19 000 as estimated by gel filtration. This crystalline inhibitor is highly active against both bovine pancreatic trypsin and alpha-chymotrypsin. Complexes of both trypsin-inhibitor and alpha-chymotrypsin-inhibitor have been isolated. The inhibitor which was derived from the dissociated trypsin-inhibitor complex was only 62% as effective as the original compound against either enzyme. In contrast, the inhibitor obtained from alpha-chymotrypsin-inhibitor complex retained its full original inhibitory activity for trypsin, but only 25% of its original activity against alpha-chymotrypsin. The dissociated inhibitor from alpha-chymotrypsin-inhibitor compex, despite its full inhibitory activity, had been modified to such an extent that it could no longer form any precipitable complex with trypsin. The crystalline protein-proteinase inhibitor is not homogeneous and has been resolved into two distinct inhibitors in terms of their physical and chemical properties. These two inhibitors are designated as Pinto bean proteinase inhibitor I and II and their respective minimum molecular weights are 9100 and 10 000. They differ most strikingly in their amino acid composition in that inhibitor II is void of both valine and methionine.     

CRYSTALLINE TRYPSIN : V. KINETICS OF THE DIGESTION OF PROTEINS WITH CRUDE AND CRYSTALLINE TRYPSIN

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.     

EFFECT OF THE FORMATION OF INERT PROTEIN ON THE KINETICS OF THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN

A solution of crystalline trypsinogen in dilute buffer containing a trace of active trypsin when allowed to stand at pH 5.0–9.0 and 5°C. is gradually transformed partly into trypsin protein and partly into an inert protein which can no longer be changed into trypsin either by enterokinase or mold kinase. During the process of formation of trypsin and inert protein the ratio of the concentrations of the two products in any reaction mixture remains constant and is independent of the original concentration of trypsinogen protein. This ratio varies, however, with the pH of the solution, the proportion of trypsin formed being greater in the acid range of pH. The experimental curves for the rate of formation of trypsin, as well as for the rate of formation of inert protein are symmetrical S shaped curves closely resembling those of simple autocatalytic reactions. The kinetics of formation of trypsin and inert protein can be explained quantitatively on the theoretical assumptions that both reactions are of the simple unimolecular type, that in each case the reaction is catalyzed by trypsin, and that the rate of formation of each of the products is proportional to the concentration of trypsin as well as to the concentration of trypsinogen in solution.     

Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.     

CRYSTALLINE CHYMO-TRYPSIN AND CHYMO-TRYPSINOGEN : I. ISOLATION,CRYSTALLIZATION, AND GENERAL PROPERTIES OF A NEW PROTEOLYTIC ENZYME AND ITS PRECURSOR

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.     

Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment.

Side-chain torsional potentials in the bovine pancreatic trypsin inhibitor are calculated from empirical energy functions by use of the known X-ray structure of the protein and the rigid-geometry mapping technique. The potentials are analyzed to determine the roles and relative importance of contributions from the dipeptide backbone, the protein, and the crystalline environment of solvent and other protein molecules. The structural characteristics of the side chains determine two major patterns of energy surfaces, E(X1,X2): a gamma-branched pattern and a pattern for longer, straight side chains (Arg, Lys, Glu, and Met). Most of the dipeptide potential curves and surfaces have a local minimum corresponding to the side-chain torsional angles in the X-ray structure. Addition of the protein forces sharpens and/or selects from these minima, providing very good agreement with the experimental conformation for most side chains at the surface or in the core of the protein. Inclusion of the crystalline environment produces still better results, especially for the side chains extending away from the protein. The results are discussed in terms of the details of the interactions due to the surrounding, calculated solvent-accessibility figures and the temperature factors derived from the crystallographic refinement of the pancreatic trypsin inhibitor.     

Occurrence of Antimicrobial Serin-Proteinases in Sunflower Seeds

Using an experimental approach directed to the isolation of antimicrobial proteins, we have detected the presence of a trypsin inhibitor (TI) with associated antifungal activity in sunflower seeds. Purification of the isolated protein by affinity chromatography on a trypsin‐agarose matrix confirmed that a trypsin inhibitor was responsible for the inhibition of spore germination of the fungal pathogen Sclerotinia sclerotiorum. The protein is a potent antifungal compound as it can completely inhibit the germination of S. sclerotiorum ascospores at a concentration of 14 μg/ml. The putative contribution of this TI to control fungal invasion is discussed.     

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.     

A study of the lysyl residues in the basic pancreatic trypsin inhibitor using 1H nuclear magnetic resonance at 360 Mhz.

Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Nepsilon-dimethyllsysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine-41 is involved in an intramolecular interaction with tyrosine-10. Since in the single crystal structure tyrosine-10 is involved in an intermolecular interaction with arginine-42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.     

Estimation of parameters in a multi-affinity-state model for haemoglobin from oxygen binding data in whole blood and in concentrated haemoglobin solutions.

The reduced fragment of pancreatic trypsin inhibitor lacking the six C-terminal residues, which is produced by cyanogen bromide cleavage, formed a seemingly random mixture of disulphide bonds under refolding conditions where normal pancreatic trypsin inhibitor refolds correctly and quantitatively. This illustrates the importance of the C-terminal residues in folding of the normal protein, the uniqueness of the normal folded conformation, and the apparently central role in protein folding of long-range interactions between residues distant in the primary structure.The intact polypeptide chain of reduced pancreatic trypsin inhibitor in which the methionine residue normally at position 52 had been converted to homoserine refolded slightly less readily than the normal reduced compound. This was observed to be due to an altered spectrum of single-disulphide intermediates: the normally predominant intermediate with the 30–51 disulphide bond was less stable by about 0.8 kcal/mol relative to the other normal single-disulphide intermediates. The other steps in refolding appeared to be normal, although the refolded protein was observed to be susceptible to an unexplained reaction with iodoacetate.     

A STUDY OF THE EQUILIBRIUM BETWEEN THE SO CALLED "ANTITRYPSIN" OF THE BLOOD AND TRYPSIN

1. The retarding effect of plasma on the action of trypsin can be measured quantitatively. 2. The nature of the reaction involved in effecting the retardation has been subjected to an experimental study. 3. Evidence is presented which indicates that the equilibrium between the inhibitive agent and trypsin is reached practically instantaneously and is rapidly and completely reversible. 4. This equilibrium has been studied by experiments in which we have observed (1) the effect of adding increasing amounts of plasma to a constant amount of trypsin, (2) the effect of varying the amount of trypsin while the plasma was constant, (3) the effect of dilution on the trypsin-plasma mixture. 5. The results of these experiments are discussed and it is stated that they are in quantitative agreement with the law of mass action. 6. An equation was found which fits the curves for the experiments mentioned in (4). This equation was developed from the assumption that 1 molecule of trypsin combined with 1 molecule of inhibitor to form 1 molecule of trypsin-inhibitor compound. The agreement between the results calculated by this equation and the observed results is satisfactory. It is pointed out that the equation contains two arbitrary constants and the bearing this fact may have on the calculated results is discussed. 7. We conclude from the results of our study that we have adduced evidence which suggests the following statement regarding the so called "antitryptic" property of blood. The inhibitive agent and trypsin combine to form an inactive but dissociable compound. The reaction in equilibrium is expressed by the equation Trypsin + inhibitor ⇌ trypsin-inhibitor The conditions of equilibrium are apparently governed by the law of mass action. The behavior of the equilibrium is therefore similar to the behavior of other equilibria between different inhibitive agents and enzymes discussed in the paper.     

胰蛋白酶抑制物对尼罗非鲫生长的影响

本实验研究了生、熟大豆中含有的胰蛋白酶抑制物(TI)对尼罗非鲫生长的影响。实验结果表明:沸水热处理不仅使生大豆中81.4％的TI失去活性,而且还显著地提高了大豆蛋白质系数,PER从1.12提高到1.76。当颗粒饲料的Tl含量低于0.9mg/g时,尼罗非鲫的生长正常;但高于此量吋,其生长速度明显降低。       

Determination and comparative analysis of the conformation of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from the data of two-dimensional 1H-NMR spectroscopy

On the basis of joint consideration of distance dependences between amide proton NH and protons C alpha H, NH, C beta H of the preceding in amino acid sequence residue from the torsion angles phi psi, chi 1, the correlation diagram of these proton-proton distances with the regions of sterically allowed conformational space (phi, psi) is presented and the method for the determination of the L-amino acid residues backbone conformations is proposed. The diagram was used for the determination of backbone conformations of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from Dendroaspis polylepis using the data from two-dimensional 1H-NMR spectroscopy. The analysis of backbone conformations was carried out. The individual elements of these protein molecules secondary structure were characterized and their high conformational homology was shown. The inference about qualitative coincidence of three protein molecules conformation in solution, preservation of secondary structure basic elements and their similarity with bovine pancreatic trypsin inhibitor crystalline structure was made.     

Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : II. PHYSIOLOGICAL SIGNIFICANCE. THE INFLUENCE OF PURIFIED TRYPSIN INHIBITOR ON THE COAGULATION OF THE BLOOD

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.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

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 Q _5–15 = 1.70 to Q _25–30 = 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 Q ₁₀ = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.     

Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of alpha-macroglobulin in Biomphalaria glabrata.

1. A methylamine-sensitive inhibitor was present in the plasma of B. glabrata. 2. This inhibitor decreased trypsin activity against a protein substrate, however trypsin retained activity against a low molecular weight substrate in the presence of the inhibitor. 3. Snail plasma protected trypsin from inhibition by soybean trypsin inhibitor. 4. The results give evidence for an alpha-macroglobulin proteinase inhibitor in the plasma of this gastropod mollusc.     

INACTIVATION OF CRYSTALLINE TRYPSIN

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.