The esterolytic specificity of bovine thrombin and Factor Xa.

Steady state kinetics are compared for the hydrolysis of t-butoxycarbonyl-L-lysine methyl ester and several peptidyl lysine methyl esters catalysed by bovine thrombin and Factor Xa. Thrombin-catalysed reactions have lower Km values and higher kcat/Km values than do reactions catalysed by Factor Xa. Values of kcat are comparable and do not show any particular trend. The best substrate in the present series was t-butoxycarbonylglycylglycyl-L-lysine methyl ester. Thrombin and Factor Xa may possess a hydrophobic region near the P2 binding site which is unfavourable for either asparagine or D-alanine but which readily accommodates glycine, L-alanine or L-phenylalanine. The major improvement in Factor Xa hydrolysis occurred with the occupation of the P2 site by an amino residue while for thrombin the major improvement occurred with the occupation of the P3 site.

The hydrolysis of chemically modified proteins by Factor Xa and thrombin.

Bovine thrombin and Factor Xa were shown to hydrolyse slowly several chemically modified proteins. Both enzymes hydrolyse the proteins at trypsin-susceptible bonds, with arginine, lysine or the synthetically generated S-(beta-aminoethyl)cysteine at the P1 position. Both enzymes, however, cleave at far fewer sites than trypsin. The presence of highly polar groups in the P2 position appears to hinder hydrolysis by Factor Xa or thrombin. The presence of hydrophobic or neutral amino acids around this site may make the site more susceptible to hydrolysis. Differences in the hydrolysis patterns between thrombin and Factor Xa are observed.

Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand.

The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed.

The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides.

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.

The degradation of bovine and human prothrombin by human polymorphonuclear leukocyte cathepsin G.

Cathepsin G, isolated from human polymorphonuclear leukocytes, was found to effect rapid and specific degradation and biological inactivation of bovine and human prothrombin in the absence of calcium ions with the formation of two peptide fragments from the N-terminal end of the molecule. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular weights of the fragments were 5,000 and 17,500. Proteolysis of prothrombin by cathepsin G was inhibited by calcium ions. Leukocyte proteinases such as cathepsin G may be responsible for haemorrhagic disorders associated with myelocytic leukaemia and septicaemia.

Proteolysis of bovine and human prothrombin and of bovine factor X by rat mast cell proteinase.

The alpha-chymotrypsin-like proteinase from rat peritoneal mast cells (RMCP I) rapidly destroyed the normal clotting activity of purified, calcium-free, bovine prothrombin, human prothrombin and bovine factor X and simultaneously removed similar N-terminal peptides (Mr approximately 5,000) from both prothrombin and the 'light' chain of factor X. The amino acid composition of the peptides agreed with the known composition of the regions of the respective parent molecules where gamma-carboxyglutamic acid residues are situated. Ca2+ ions protected each of the proteins from proteolysis and loss of procoagulant activity. Prolonged incubation in the standard physiological assay medium used for prothrombin or treatment with Echis carinatus venom indicated that the thrombogenic portion of prothrombin survived proteolysis by RMCP I. This restricted proteolysis was confirmed by electrophoretic analysis.

The behaviour of trypsin towards alpha-N-methyl-alpha-N-toluene-p-sulfonyl-L-lysine beta-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin.

1. alpha-N-Methyl-alpha-N-toluene-p-sulphonyl-l-lysine beta-naphthyl ester (MTLNE) was synthesized as its hydrobromide and shown to be slowly hydrolysed by bovine pancreatic trypsin. The acylation step, however, is so much faster than deacylation of the acyl-enzyme that spectrophotometric measurement of the ;burst' of beta-naphthol provides a convenient method for determining the absolute molarity of trypsin solutions. 2. By using the same stock solution of trypsin, application of this method at pH4.0 and pH7.0 as well as that of Bender et al. (1966) at pH3.7 gave concordant results. 3. Provided that [S](0)>[E](0), the size of the ;burst' is independent of substrate concentration. 4. In the trypsin-catalysed hydrolysis of alpha-N-toluene-p-sulphonyl-l-arginine methyl ester, MTLNE functions as a powerful non-competitive inhibitor. 5. There is no detectable reaction between MTLNE and either bovine pancreatic alpha-chymotrypsin at pH4.0 or bovine thrombin at pH6.0.

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.

A radiochemical titrant for the determination of the operational molarity of solutions of acid proteinases.

N-Diazoacetyl-L-phenylalanine 3-phenyl[2,3-3H]propylamide was synthesized and shown to inhibit pepsin A (EC3,4,23.1) and cathepsin D (EC 3.4.23.5) irreversibly and stoicheiometrically in the presence of Cu2+. Quantitative separation of the inhibited enzyme from excess reagent by gel filtration followed by measurement of the radioactivity of the protein peak provided a method for determining the operational molarity of these enzymes. Several other putative active-site-directed irreversible inhibitors were synthesized, but were inactive. Data on the synthesis of these compounds have been deposited as Supplementary Publication SUP50096 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

The irreversible inhibition of urokinase, kidney-cell plasminogen activator, plasmin and beta-trypsin by 1-(N-6-amino-n-hexyl)carbamoylimidazole.

1-(N-Amino-n-hexyl)carbamoylimidazole hydrochloride was synthesized and shown to be a potent irreversible inhibitor of human urokinase (EC 3.4.21.31), pig kidney-cell plasminogen activator (EC 3.4.21.-), human plasmin (EC 3.4.21.7) and bovine pancreatic beta-trypsin (EC 3.4.21.4). The kinetics of inhibition of the enzymes were determined by monitoring the hydrolysis of an appropriate fluorogenic substrate. Bovine thrombin and Factor Xa are hardly affected by the inhibitor.

Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine alpha- and beta-trypsin.

Several esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid and alpha-N-toluene-p-sulphonyl-l-homoarginine by alpha- and beta-trypsin were compared. On the basis of values of the specificity constants (k(cat.)/K(m)), the two enzymes display similar catalytic efficiency towards some substrates. In other cases alpha-trypsin is less efficient than beta-trypsin. It is possible that alpha-trypsin possesses greater molecular flexibility than beta-trypsin.