Characterization of azidobenzamidines as photoaffinity labels for trypsin

Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversible in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-l-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation. Maximum inactivation of trypsin is achieved at pH 6.2 using either azidobenzamidine derivative. Evaluation of the pH dependence of photoaffinity labeling may provide a sensitive tool for probing conformational changes in inhibitor binding sites. These studies provide a basis for the use of azidobenzamidines as photoaffinity analogs of lysine and arginine side chains.     

Inhibition of Monoamine Oxidase by Phenyl Azides

We had previously shown that 4-fluoro-3-nitrophenyl azide (FNPA) is a competitive inhibitor of both types of monoamine oxidase (MAO) in the dark, but it is a preferential photoaffinity label for only the type B MAO (MAO-B). Recently we synthesized a number of arylazido compounds with structures related to FNPA and determined the effects of these compounds on the two types of MAO in rat brain cortex. We found that the fluoro group of FNPA was not required for the inhibition of MAO activities because neither the presence nor the position of the fluoro group affected its inhibition of MAO. On the other hand, both the nitro and the azido groups of FNPA were shown to be important for FNPA inactivation of two types of MAO. The inhibitory potency was significantly lower for compounds without either group. Furthermore, we found that all nitrophenyl azide isomers except 2-nitrophenyl azide were photodependent inhibitors of MAO-B. Under the same experimental conditions none of the compounds photoinactivated MAO-A. On the basis of these findings, mechanisms for FNPA inhibition of the two types of MAO are discussed.     

4-Fluoro-3-nitrophenyl azide binding sites on purified beef liver monoamine oxidase B

Previous work from this laboratory has shown that 4-fluoro-3-nitrophenyl azide (FNPA) is an effective photoaffinity labeling probe for MAO-B (Chen et al., Biochem. Pharmac.34, 781–785, 1985). The FNPA binding sites have been further studied by using [³H]FNPA. When [³H]FNPA was photolyzed with purified beef liver MAO, then subjected to tryptic and chymotryptic digestion, three radioactive peaks were observed after Sephadex G-25 column chromatography procedure. The extent of [³H]FNPA incorporation varied directly with [³H]FNPA concentration. They could be protected by the presence of the substrate (phenylethylamine) or inhibitors (pargyline and trans-phenylcyclopropylamine) of MAO-B during photolysis. These protections were concentration dependent. Furthermore, the decrease in [³H]FNPA labeling in the presence of inhibitors paralleled the decrease in MAO catalytic activity. These results suggest that the FNPA binding sites were related to the active site of MAO-B. Under the same conditions, the separation profiles of [³H]FNPA labeled and [³H]pargyline labeled tryptic-chymotryptic peptides after Sephadex G-25 column chromatography are distinctly different. This result suggests that FNPA labeling sites may be different from the pargyline binding site. Since pargyline binds to the prosthetic group(-FAD) of MAO, [³H]FNPA may label different domains of the active site. This probe may be useful for the characterization of the active site of MAO-B.     

Photolytic inhibition and labeling of proteins with aryl diazonium compounds.

In the course of preparing aryl azide derivatives for use as photoprobes, we have observed significant light sensitivity in the precursor aryl diazonium compounds. The photosensitive properties of this class of compounds are of interest since they will seek out cationic binding sites in biological targets, and can be employed to inhibit complementary targets at acid pH. The relationship between photolytic change in the structure of diazonium compounds and the corresponding change in function of a biological target are presented. Experiments are described in which the dark and light sensitive properties of a model diazonium compound, diazobenzene sulfonate (DBS), were determined. The ultraviolet spectra were used to evaluate the dark stability and light sensitivity of DBS. Chymotrypsin and trypsin served as functioning targets for further evaluation of the photochemical properties. Both enzymes are stable to the probe in the dark at acid pH. A rapid loss of enzyme activity was observed following flash photolysis of DBS-enzyme solutions. Photolytic incorporation of radioactive DBS into chymotrypsin was observed. Aryl diazonium salts can be employed to probe the availability of complementary sites in biological targets at different acid pH values.     

Inactivation of Enzymes by Linoleic Acid Hydroperoxides and Linoleic Acid

The inactivation of the enzymes by linoleic acid hydroperoxides (LAHPO) was tested in connection with the toxicity of oxidized fat. At the same time, the inhibition of enzyme activities by linoleic acid was also tested. Ribonuclease (RNase), trypsin, chymotrypsin and pepsin which are considered to be simple proteins and not to be SH-enzymes were chosen as the enzymes. RNase was largely inhibited by LAHPO, but the other enzymes were inhibited by linoleic acid as well as LAHPO. The inhibition of each enzyme occurred at different pH. This fact may show that the inhibition occurs by binding of such hydrophobic compounds to the enzyme, and that the surface exposition of hydrophobic region may depend on the pH. Not only the reaction of some specific amino acid residue in the protein molecules with LAHPO, but also the binding of these hydrophobic compounds must be remembered in the mechanism of inhibition.     

A simple method to determine trypsin and chymotrypsin inhibitory activity

A colorimetric method for serine protease inhibition was modified using N-Acetyl-DL-Phenylalanine beta-Naphthylester (APNE) as the substrate and o-Dianisidine tetrazotized (oD) as the dye. The reaction generated a single peak absorbing at 530 nm for both trypsin and chymotrypsin. Standard curves with increasing enzyme concentrations showed strong linearity. A standard curve for the serine protease inhibitor, Bowman-Birk Inhibitor (BBI), has been made using this modified method. The IC50 for 3 U of trypsin was found to be 33 ng and the IC50 obtained for 3 mU of chymotrypsin was 53 ng. A recombinant BBI (rBBI) gene was constructed, cloned and expressed in the yeast Pichia pastoris. Evaluating samples of rBBI for protease inhibitory activity by the gel activity method failed to quantify the inhibitor amounts, due to high sensitivity for trypsin inhibition and low sensitivity for chymotrypsin inhibition. After development, the results could not be quantified, even to the extent that 1 microl of rBBI could not be detected with chymotrypsin inhibition. Therefore, a modified method for trypsin and chymotrypsin inhibition was used to evaluate the level of rBBI-expression for these same samples. The level of rBBI expression was calculated to be 50-56 ng/microl of media. These amounts fit into the range of values previously obtained by Western blot analysis. This modified method allows us to combine the sensitivity of the gel activity method with the quantification attributes of a Western blot. Thus, the modified method represents a significant improvement in speed, sensitivity and reproducibility over the gel activity method.     

Phenothiazine-N-carbonyl chloride, a specific inactivator of chymotrypsin

Phenothiazine-N-carbonyl chloride inactivated chymotrypsin and trypsin by means of a 1:1 stoicheiometric reaction. Its reaction with chymotrypsin was 29 times as fast as that with trypsin and was inhibited by indole. The reaction of phenothiazine-N-carbonyl chloride with chymotrypsin resembled an enzyme-substrate reaction in which the deacylation step is rate-limiting. Slow deacylation occurred, resulting in complete regeneration of active enzyme in 15h. The pH-rate profile of the inactivation process had a maximum at pH7.8. These data and other evidence indicate that the reaction of phenothiazine-N-carbonyl chloride with chymotrypsin exhibits ;kinetic specificity'. Therefore any hypothesis that attempts to describe the topography of the active site of chymotrypsin should take into account the reactivity of phenothiazine-N-carbonyl chloride. The above findings, as well as recent reports of others, are examined within the context of a hypothesis given in an earlier paper (Erlanger, 1967).     

Recovery of aprotinin from insulin industrial process effluent by affinity adsorption

Aprotinin is a protease inhibitor found in bovine organs and used as a valuable human therapeutic compound. In this work, a process for the recovery of aprotinin from insulin industrial process effluent via affinity adsorption on immobilized trypsin and chymotrypsin was developed. First, process conditions were set as a result of a study of the effects of pH and ionic strength on pure aprotinin adsorption and desorption utilizing an experimental design methodology. The best conditions obtained with immobilized trypsin as the ligand were adsorption at 0.018 M NaCl and pH 8.7 and desorption at 0.018 M NaCl and pH 2.1. For immobilized chymotrypsin, the best conditions were adsorption at 0.582 M NaCl and pH 8.0 and desorption at 0.582 M NaCl and pH 2.1. Recovery of the inhibitor from the effluent was carried out utilizing a two-step process: trypsin-agarose adsorption followed by chymotrypsin-agarose adsorption. Analysis of the chromatographic fractions by trypsin and chymotrypsin inhibition and capillary electrophoresis assays strongly suggested that the recovered inhibitor is aprotinin.     

Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and photosystem II.

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.     

Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition.

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.     

Gamma-irradiated chymotrypsin-like proteins. II. Connection between inactivation and structural changes.

The radiation yeilds of unfolding (Gconf) determined by the method of tryptophan fluorescence coincide with the radiation yields of proteolytic inactivation (Gin) for chymotrypsin-like (CT-like) enzymes on irradiation in air, both in solution and in the dry state with futher dissolution at pH7. It can be supposed that the unfolding is the main process determining the proteolytic gamma-inactivation of CT-like enzymes. It was also shown that the transition of chymotrypsin and trypsin gamma-irradiated at acid pH to neutral pH is an additional action, leading to unfolding of part of the molecules.     

Protease inhibitors from ripened and unripened bananas.

The proteolysis of casein by trypsin, chymotrypsin and papain was inhibited by ripened and unripened bontha, poovan, nendran, cavendish and rasthali bananas. The inhibition of trypsin, chymotrypsin and papain by different ripened banana cultivars was much more than that of unripened banana cultivars. The trypsin and chymotrypsin inhibitory activity of ripened poovan was heat stable, resistant to pronase and partly stable to trypsin but the trypsin and chymotrypsin inhibitory activity of unripened poovan was stable to heat and resistant to pronase only. The partial stability of trypsin inhibitory activity and instability of papain inhibitory activity of ripened poovan to alkaline pH suggests that the inhibitory factors of trypsin and papain were dissimilar. The probable role of unripened banana papain inhibitors in curing stomach ulcers and antinutritional role of ripened banana trypsin inhibitors is discussed.     

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers.

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.     

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

1. p-Nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate (NPABC) was synthesized and shown to acylate alpha-chymotrypsin stoicheiometrically; reaction at 25 degrees occurs almost instantaneously at pH7.04 and within 2min. at pH5.04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of alpha-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5.04 and pH7.04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than alpha-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7.04. At pH5.04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for alpha-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of alpha-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when alpha-chymotrypsin and trypsin are acylated by NPABC.     

Mechanism of inactivation of trypsin by antithrombin

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

Glycation improves the thermostability of trypsin and chymotrypsin

A novel glycation procedure, in vacuo glycation, was used to attach glucose covalently to the lysine residues of trypsin and chymotrypsin. Glycated trypsin and glycated chymotrypsin have greatly increased thermostability compared to the native enzymes. For example, glycated bovine trypsin, incubated at 50 degrees C and pH 8.0 for 3 h, retained more than 50% of its original activity whereas the native enzyme was inactivated under the same conditions. Similarly, after incubation at 50 degrees C and pH 8.0, glycated bovine chymotrypsin retained 45% of its original activity and the native enzyme was inactivated. Glycated porcine trypsin is exceptionally thermostable and could be used to digest native ribonuclease at 70 degrees C without the need for prior denaturation. The apparent increase in the thermal stability of the glycated proteins observed in activity measurements is also reflected by an increase in the T(m) values determined with differential scanning calorimetry (DSC) and circular dichroism (CD). The glycation does not alter the activity or specificity of these enzymes.     

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).