Trypsin Molecules Research Articles

Purification and properties of rat α 2 acute-phase macroglobulin

α 2 acute-phase macroglobulin was isolated from plasma of turpentine-injected rats. In the method conditions known to damage the biological activities of α 2 macroglobulin are avoided. The procedure successively involves: rivanol precipitation, concanavalin A-Sepharose chromatography and ion-exchange chromatography on DEAE-cellulose. Proteolytic activities were minimized throughout the purification. Thus α 2 macroglobulin was obatined in a 20% yield and was pure by biochemical and immunological criteria. Its molecular weight appeared to be 760 000 and it consisted of four subunits ( M r 190 000). The protein has an A 1 cm 1% = 8.8 and an isoelectric point = 4.8. The amino acid and carbohydrate compositions were determined. Our preparations bound 1 molecule of trypsin or 1 molecule of plasmin/molecule of α 2 macroglobulin. Kinetic parameters for α 2 macroglobulin-bound trypsin and plasmin were determined and compared with those of free trypsin and plasmin using butoxycarbonyl- l-valylglycyl- l-arginine-2-naphthylamide and benzoyl- l-arginine ethylester as substrates.

Use of energy transfer to assay the asseciation of proteins with lipid membranes

A method is described for the rapid and quantitative assay of the association of proteins with lipid membranes. The method utilizes the nonradiative transfer of energy between fluorescent groups in the protein and fluorescent groups attached to the polar heads of the lipids in the membrane. The association constant for the electrostatic association of trypsin with bilayers of the negatively charged lipid, 1,2-dimyristoyl- sn-glycerol-3-phosphoric acid methyl ester (MPA) at a salt concentration of 5 m m and pH 8 was found to be (2.32 ± 0.07) × 10 5 m −1. The stoichiometry of lipid to protein in the association complex is 40–50 lipid molecules per trypsin molecule. Honey-bee venom melittin and the serum apolipoprotein, apo-Lp-Ala, were shown to associate with lecithin vesicles. Acetylcholine esterase isolated from the electric organ of the electric eel according to the method of Leuzinger and Baker [(1967) Proc. Nat. Acad. Sci. USA 57, 446–451] was shown to be incapable of associating with lecithin or MPA bilayers.

Human pancreatic cationic trypsin as a possible mediator of pancreatic shock as inferred from a study of its interaction with serum and α2-macroglobulin

1. (1) The study of the interaction between human pancreatic proteases and human serum shows that cationic trypsin binds preferentially to α 2-macroglobulin, whereas chymotrypsin II binds preferentially to α 1-proteinase inhibitor. 2. (2) One molecule of α 2-macroglobulin is able to combine with two molecules of cationic trypsin to form a complex which is 80, 10, and 0.17% active on benzoyl- dl-arginine- p-nitroanilide, casein, and chymotrypsinogen, respectively. 3. (3) From these results it is inferred that cationic trypsin may be a major mediator of the shock observed during acute pancreatitis.

Tryptic hydrolysis at asparagine residues in globin chains

Commercially available Tos-PheCH 2Cl-treated or untreated bovine trypsin (EC 3.4.21.4) is shown to catalyse minor tryptic hydrolysis at the carboxyl side of asparagine residues in globin chains. This activity is not removed by the purification of enzyme, using CM-cellulose chromatography and subsequent affinity chromatolography on trypsin inhibitor columns, neither is it inhibited by Tos-PheCh 2Cl treatment of the CM-cellulose purified enzyme. It is suggested that the ability to hydrolyse globin chains at asparagine residues may represent an inherent feature of the trypsin molecule.

Preparation and characterization of a dextran-trypsin conjugate.

Bovine pancreatic trypsin was coupled to dextran after activation of the polysaccharide by cyanogen bromide. The soluble dextran-trypsin conjugated was purified by molecular sieve chromatography. After coupling, 53% of the esterase activity of trypsin remained, but the conjugate had only 7% of the caseinolytic activity of the native enzyme. The modified trypsin showed greater resistance than the native enzyme to inactivation by heat treatment, autodigestion, or denaturing agents, and was also more resistant to inhibition by trypsin inhibitors, particularly ovomucoid. Treatment with dextranase partly removed the improved stability properties and resistance to inhibition of the trypsin-dextran conjugate. The conjugated enzyme preparation consists of a heterogenous mixture of macromolecular aggregates, each containing many trypsin and many dextran molecules linked together. Intramolecular cross-linking of enzyme molecules by polysaccharide chains is considered to be responsible for stabilization of the tertiary structure of the enzyme molecules in the conjugate.

Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.

35] Immobilization as a means of investigating the acquisition of tertiary structure in chymotrypsinogen

Publisher Summary This chapter discusses the immobilization as a means of investigating the acquisition of tertiary structure in chymotrypsinogen. Immobilization of chymotrypsinogen through covalent attachment to an insoluble matrix provided the key for investigation of the regeneration of biologically functional tertiary structure from completely reduced, denatured zymogen chains under conditions that would minimize interactions leading to nonspecific aggregation and formation of intermolecular disulfide bridges. Studies of reduction and reoxidation of immobilized trypsin and ribonuclease and studies of activation of immobilized pepsinogen are also presented in the chapter. It is found that the three-dimensional conformation of at least the catalytically active portion of the trypsin molecule is determined by the amino acid sequence alone. It is also reported that the amino acid composition of Sepharose-pepsinogen preparations is not altered by exposure to pH 2.

The refined crystal structure of bovine β-trypsin at 1·8 Å resolution : I. Crystallization, data collection and application of patterson search techniques

X-ray intensity data to 1·8 A resolution have been collected from a single crystal of benzamidine-inhibited bovine β -trypsin at pH 7·0. Given the refined atomic co-ordinates as found in the crystal structure of bovine trypsin-pancreatic trypsin inhibitor complex (Huber et al. , 1974) the trypsin molecules have been located within the trypsin crystals applying Patterson search techniques. Using the optimal orientation and position parameters a crystallographic R -factor of 0·43 was calculated for data from 6·8 to 1·8 A resolution.

Preferred binding of bovine and porcine trypsins at two different sites on chicken ovoinhibitor. Reduced dissociation of mixed trypsin complexes.

Dissociation of mixed trypsin (bovine plus porcine trypsin) complexes with chicken ovoinhibitor was used to investigate the nonequivalence of the two binding sites for trypsin on the inhibitor. Previous work has shown that 1 mol of trypsin dissociates much more rapidly than the 2nd from unmixed trypsin complexes, those containing 2 mol of one kind of trypsin, bovine or porcine, per mol of inhibitor. However, only approximately 0.5 to 0.6 mol of trypsin dissociated in the rapid step from the mixed trypsin complexes, those containing 1 mol each of bovine and porcine trypsin. Rates of the slow dissociation steps for the two types of complexes did not differ appreciably from each other. A general dissociation scheme is proposed, in which each of the 2:1 complexes can lose a trypsin molecule from either in two parrallel first order reactions, producing two different 1:1 complexes, which subsequently dissociate to yield free ovoinhibitor and a second trypsin molecule. In this scheme, both the earlier results with unmixed trypsin complexes and the preponderance (approximately 3:1) of slow dissociation from the mixed trypsin complexes can be rationalized if bovine trypsin is retained preferentially at one of the two trypsin binding sites on chicken ovoinhibitor, and porcine trypsin at the other. That is, one site allows rapid dissociation of porcine trypsin and slow dissociation of bovine trypsin, whereas the other allows rapid dissociation of bovine trypsin and slow dissociation of porcine trypsin.

The partial amino acid sequence of trypsin inhibitor II from garden bean, Phaseolus vulgaris, with location of the trypsin and elastase-reactive sites

The amino acid sequences of garden bean (Phaseolus vulgaris) trypsin inhibitor II and a related molecular species II' have been examined. The entire sequence of II' has been determined with the exception of five internal residues. The garden bean inhibitors are highly homologous to the Bowman-Birk soybean inhibitor and lima bean trypsin inhibitor IV. The trypsin-reactive site has been located in the second half of the molecule, while the first reactive site has been found to be directed against elastase. Garden bean inhibitor II (and II') is thus a double-headed inhibitor, simultaneously inhibiting 1 molecule of trypsin and 1 of elastase.

Evidence That the Two Binding Sites for Trypsin on Chicken Ovoinhibitor Are Not Equivalent: DISSOCIATION OF THE COMPLEXES WITH PORCINE TRYPSIN

Abstract Complexes between chicken ovoinhibitor (OI) and porcine trypsin were allowed to dissociate in the presence of the active site titrant, p-nitrophenyl p'-guanidinobenzoate, which reacts extremely rapidly with free active trypsin (T). The rate of the slower, postburst reaction with titrant, which is a measure of the rate of dissociation of the complexes, showed a distinct break not observed previously with bovine trypsin (Zahnley, J. C., and Davis, J. G. (1970) Biochemistry 9, 1428–1433). The OI-trypsin complexes dissociate according to the scheme OI-T2 ⇌ OI-T ⇌ OI + T + T With porcine trypsin, rate constants for dissociation of the OI-T2 and OI-T complexes were 7.5 x 10-2 and 4 to 8 x 10-4 s-1, respectively. The OI-T2 complex obtained with porcine trypsin dissociated 6 to 8 times faster than that obtained with bovine trypsin, perhaps reflecting structural differences between the two trypsins. The greater ratio of dissociation rate constants, kd, with porcine trypsin (g 100, against 9 for bovine trypsin) shows clearly that the two binding sites for trypsin on the single polypeptide chain of chicken ovoinhibitor are not equivalent. This appears to be due to intrinsic differences between the two sites, rather than to changes induced by the binding of the first trypsin molecule. An association rate constant, ka, of 8 x 104 m-1 s-1 for formation of the OI-bovine trypsin (1:1) complex was calculated from the inhibition constant (Ki) and the kd. This value of ka is lower than those previously reported for other naturally occurring trypsin inhibitors.

Trypsin inhibition of electron transport

1. 1. A relaxation spectrophotometer was employed to measure the effects of trypsin treatment on electron transport in both cyclic and non-cyclic chloroplast reactions. The parameters measured were electron flow rate through P700 (flux) and the time constant for dark reduction of P700. 2. 2. In the reduction of methyl viologen by the ascorbate-2,6-dichlorophenol-indophenol (DCIP) donor couple, there was no effect of trypsin on P700 flux or on the time constant for dark reduction of P700. In the phenazine methosulfate (PMS) cyclic system, trypsin had either a slightly stimulatory or slightly inhibitory effect on the P700 flux, depending on the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU): either effect being marginal compared to trypsin effects on Photosystem II. With both ferricyanide and methyl viologen reduction from water, trypsin treament gave a first order decline in P700 flux: which matched the trypsin-induced decline in electron transport with the water to DCIP system, measured by dye reduction. This implies that Photosystem II is inhibited. The inhibition of Photosystem II was up to 90% with a 6–10-min trypsin treatment. This result is consistent with the concept of Photosystem I (P700) being in series with Photosystem II in the electron transfer sequence. 3. 3. Cyclic phosphorylation was severely inhibited (85%) by trypsin treatment which had a somewhat stimulatory effect on P700 flux, indicating uncoupling. Non-cyclic phosphorylation was uncoupled as well as electron flow being inhibited since the P 2e ratio decreased more rapidly as a function of trypsin incubation time than inhibition of electron flow. The two effects, uncoupling and non-cyclic electron flow inhibition, are separate actions of trypsin. It is probably that the uncoupling action of trypsin is due to attack on the coupling factor protein, known to be exposed on the outer surface of thylakoids. 4. 4. Trypsin treatment caused an increase in the rate constant, k d, for the dark H + efflux, resulting in a decreased steady state level of proton accumulation. The increased proton efflux and the inhibition of phosphorylation are consistent with an uncoupling effect on trypsin. 5. 5. Trypsin treatment did not reduce the manganese content of chloroplasts: as reported by others, Tris washing did remove about 30% of the chloroplast manganese. 6. 6. Electron micrographs of both negatively stained and thin-sectioned preparations showed that, under these conditions, trypsin does not cause a general breakdown of chloroplast lamellae. Inhibition by trypsin must therefore result from attacks on a few specific sites. 7. 7. Both System II inhibition and uncoupling occur rapidly when trypsin treatment is carried out in dilute buffer, a condition which leads to thylakoid unstacking, but both are prevented by the presence of 0.3 M sucrose and 0.1 M KCl, a condition that helps maintain stacked thylakoids. Evidently vulnerability to trypsin requires separation of thylakoids. 8. 8. Since trypsin does not appear to disrupt thylakoids nor prevent their normal aggregation in high sucrose-salt medium and since the trypsin molecule is probably impermeable, it is probable that the site(s) of trypsin attack in System II are exposed on the outer thylakoid surface.

Identification of Essential Carboxyl Groups in the Specific Binding Site of Bovine Trypsin by Chemical Modification

Abstract The carboxyl groups essential for the enzyme activity of bovine trypsin were labeled and identified by means of a chemical modification procedure. Chromatographically purified bovine β-trypsin, which contains a single polypeptide chain, was treated with the water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide, and glycinamide, according to the method of Koshland et al. (13–15). Coupling of the protein carboxyl groups with glycinamide in amide linkage resulted in nearly complete loss of the enzyme activity as determined by an esterolytic assay using Nα-benzoyl-l-arginine ethyl ester as substrate. The extent of modification of the carboxyl groups and the extent of enzyme inactivation were found to be decreased in the presence of the competitive inhibitor benzamidine, indicating that certain essential carboxyl groups were protected from modification by the inhibitor. The carboxyl groups masked by the inhibitor were then reacted, after removal of the inhibitor by dialysis, with the carbodiimide reagent and 14C-glycinamide. After enzymatic degradation of the radioactive trypsin derivative, the labeled peptides were isolated and identified by their amino acid composition. The primary site of labeling was found to be aspartyl-177, strongly suggesting that this residue provides the anionic center for specific substrate binding. Some radioactivity was also associated with Asp-182. This residue reacted very slowly with the carbodiimide reagent, but it could be readily modified in the presence of 8 m urea, indicating that it is partially buried in the molecule. In 8 m urea, another carboxyl group, Asp-90, became available to modification. The failure of this residue to react in the native state suggests that Asp-90, which is homologous with an aspartyl residue known to be buried in chymotrypsin (21), is completely buried in the trypsin molecule. The enzyme which had been modified in the absence of benzamidine and had lost 99% of its specific esterolytic activity retained its reactivity toward diisopropyl phosphorofluoridate to a significant extent, indicating that the inactivation was due primarily to loss of the specificity site. The reaction of tyrosine with the carbodiimide was not a major cause of the inactivation because the regeneration of tyrosyl residues by hydroxylamine resulted in no significant change in the activity of modified trypsin preparations.

Tyrosine and tryptophan residues and amino groups in thrombin related to enzymic activities

1. 1.|The reactivities and ionization characteristics of tyrosine and tryptophan residues and amino groups in thrombin were examined in relation to esterolytic, proteolytic and clotting activities. 2. 2.|All 9 tyrosine residues in the thrombin molecule may be classified into 3 types: (a) n = 4 with p K = 10.2 ( m = 1.0); (b) n = 2 with p K = 11.1 ( m = 3.0); and (c) n = 3 with higher p K values including slowly ionizing and non-ionizing residues. 3. 3.|Acetylimidazole reacted with the 4 tyrosine residues which are possibly identical with the first group of 4 residues having a p K = 10.2 ( m = 1.0). 4. 4.|2 of the 6 tryptophan residues in the thrombin molecule reacted with H 2O 2; its esterolytic, proteolytic or clotting activities decreased, respectively, to 60, 30 and 30% of the original level. 5. 5.|13 of the total 23 amino groups in the thrombin molecule reacted with β-naphthoquinone 4,6-disulfonic acid (NQDS-4,6), and some amino groups reactive at lower concentrations of NQDS-4,6 were closely associated with the proteolytic and clotting activities. 6. 6.|The results are discussed in comparison with the trypsin molecule which resembles thrombin in its substrate specificity.

Chemical modifications of amino groups of trypsin

Reactivity of amino groups of chromatographically homogenious β-trypsin was studied by chemical modification. Guanidination by 1-amidinyl-3, 5-dimethyl pyrazole nitrate led to modification of 10 out of 14 lysyl residues per molecule of trypsin. The guanidinating reagent was found to be a competitive inhibitor of trypsin. Extent of acetamidination by methyl acetimidate hydrochloride was affected by pH, temperature and the presence of various competitive inhibitors. All 14 ϵ-amino groups of lysyl residues could be acetamidinated in the presence of the competitive inhibitors at pH 9.5 and 25° without loss of enzyme activity. When 11 or more lysyl residues are modified by acetamidination, the enzyme is stable against autolysis in the absence of Ca ++. The enzyme with 10 of its 14 lysyl residues guanidinated loses its enzyme activity by autolysis at neutral pH at a rate comparable with unmodified enzyme.

Specific Binding of Thionine to the Active Site of Trypsin

Abstract The binding of thionine (3,6-diaminophenothiazine) to trypsin is associated with a red shift in the visible absorption spectrum of the dye. Spectrophotometric titration and equilibrium dialysis measurements are consistent with the presence of a single thionine-binding site on the trypsin molecule. At pH 7 and 4°, Kdiss for the trypsin-thionine complex is 1.2 x 10-5 m. The dye is displaced from trypsin by substrates, competitive inhibitors, and the chemically reactive substrate analogue 1-chloro-3-tosylamido-7-amino-2-heptanone. Thus, the thionine-binding site overlaps with the active site region in trypsin. The dye binds very weakly to trypsinogen, with little spectral perturbation. Thionine, at low concentrations, also binds at a single site on chymotrypsin and chymotrypsinogen, showing the same spectral characteristics and a Kdiss value of 4.5 x 10-4 m at pH 7.0 and 4°. The binding of thionine to chymotrypsin is not affected by substrates, inhibitors, or chemically reactive substrate analogues; hence, the thionine-binding site in chymotrypsin does not appear to include any part of the active site region.

Effect of adsorption on the radiation sensitivity of trypsin

Many of the properties of trypsin molecules are changed when they are adsorbed at an air-quartz or air-glass interface. For example, contrary to some radiobiological postulates, the radiation sensitivity of trypsin at these air-solid interfaces is either the same as or less than that for trypsin in solution. Also, at 228 nm the absorbance of trypsin films is 1.7 that in solution, suggesting that adsorption either causes preferential orientation of some of the transition dipoles relative to the interface or else causes conformation changes. Further, the extent of the change in inactivation yield apparently depends upon the polarization of the light relative to the interface. Thus, presumably excitation of certain electronic transitions is more effective in initiating enzymic inactivation than is excitation of others. Similar to ultraviolet effects in solution, the inactivation yields for the trypsin films measured by an assay employing hemoglobin in 5 m urea are 2.5 times as large as those utilizing only buffer and BAEE substrate. By contrast, the temperature coefficients for inactivation of adsorbed trypsin are different than those observed in solution for a comparable temperature range. The quantum yield for trypsin on the slides was not changed by rather large alterations in the relative humidity nor by flushing the samples with N 2 or air during irradiation.

The Effects of Added Agar on the Radiation Sensitivity of Trypsin in Solution

A previous study in this series was concerned with the effects of radiation on the two-component system, trypsin plus the gel-forming substance agar. In firm gels the inactivation of trypsin was greatly depressed from that observed for a comparable concentration of trypsin in solution alone. With much smaller concentrations of agar, large pH effects were observed, suggesting the occurrence of fairly specific agar-trypsin complexes (1). The present experiments were designed to investigate further the changes in trypsin radiosensitivity caused by the addition of very small amounts of agar. In particular, this report is concerned with the extent of complex formation and the effects of trypsin-agar interactions on the kinetics of inactivation of the enzyme. An important finding is that, even in such a relatively simple system, three classes of trypsin molecules with widely differing properties can be demonstrated. This raises important questions concerning the relevance of studies in model systems to an understanding of radiobiological mechanisms in cells.

DISC ELECTROPHORESIS OF IRRADIATED TRYPSIN*

Abstract— Trypsin inactivated by u.v. radiation, gamma radiation, visible radiation in the presence of sensitizing dyes, and autolysis, was examined by the method of disc electrophoresis. Untreated Worthington twice crystallized salt‐free trypsin separated into four bands which moved toward the cathode; the main band, which had the greatest mobility, contained all of the detectable tryptic activity. The next most mobile band has been assumed to be a chymotrypsin contaminant. The other two bands are of unknown nature. A progressive loss of all the bands was observed when the enzyme was inactivated by those procedures which produce a ‘damaged’ class of trypsin molecules, i.e. flavin‐sensitized photooxidation, autolysis, and treatment with u.v. and gamma radiation. No loss of the main band was observed during photoinactivation with methylene blue and eosin Y as sensitizers. In this latter case, it is postulated that the trypsin inactivation products must be of such a nature that the net charge and conformation of the protein is not greatly changed, thus permitting all of the protein to remain in the same band during electrophoresis.

THE PHOTOINACTIVATION OF TRYPSIN AS SENSITIZED BY METHYLENE BLUE AND EOSIN Y*

Abstract— The kinetics of the photoinactivation of trypsin as sensitized by methylene blue and by eosin Y were investigated. The time‐course of inactivation was first‐order. The rates of inactivation were essentially identical as measured by three methods: the casein assay, the benzoyl‐L‐arginine ethyl ester assay, and the hemoglobin (in 5 M urea) assay. This is in sharp contrast to the results of earlier studies with flavins as photosensitizers, where the rate of inactivation was found to be much greater when measured by the hemoglobin assay than when measured by the other two assays. These differences are interpreted as indicating the production of a urea‐sensitive ‘damaged class’ of trypsin molecules during photodynamic inactivation with flavins as sensitizers, but not with methylene blue and eosin Y. The results presented indicate that the mechanism of the photodynamic inactivation of enzymes might be different with different photosensitizing dyes.