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. 1968 Mar;107(1):97–102. doi: 10.1042/bj1070097

The behaviour of trypsin towards α-N-methyl-α-N-toluene-p-sulphonyl-l-lysine β-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin

D T Elmore 1, J J Smyth 1
PMCID: PMC1198615  PMID: 5689288

Abstract

1. α-N-Methyl-α-N-toluene-p-sulphonyl-l-lysine β-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 β-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 α-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 α-chymotrypsin at pH4·0 or bovine thrombin at pH6·0.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BECHET J. J., YON J. MISE EN 'EVIDENCE D'UN EFFET ALLOST'ERIQUE LORS DE L'HYDROLYSE DU P-TOLU'ENE SULFONYL-L-ARGINYL-M'ETHYL ESTER PAR LA TRYPSINE. Biochim Biophys Acta. 1964 Jul 8;89:117–126. [PubMed] [Google Scholar]
  2. Baines N. J., Baird J. B., Elmore D. T. The kinetics of hydrolysis of derivatives of arginine, homoarginine and ornithine by trypsin. Biochem J. 1964 Mar;90(3):470–476. doi: 10.1042/bj0900470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baird J. B., Curragh E. F., Elmore D. T. Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of gamma-guanidino-L-alpha-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin. Biochem J. 1965 Sep;96(3):733–738. doi: 10.1042/bj0960733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bender M. L., Begué-Cantón M. L., Blakeley R. L., Brubacher L. J., Feder J., Gunter C. R., Kézdy F. J., Killheffer J. V., Jr, Marshall T. H., Miller C. G. The determination of the concentration of hydrolytic enzyme solutions: alpha-chymotrypsin, trypsin, papain, elastase, subtilisin, and acetylcholinesterase. J Am Chem Soc. 1966 Dec 20;88(24):5890–5913. doi: 10.1021/ja00976a034. [DOI] [PubMed] [Google Scholar]
  5. Creaser E. H., Bennett D. J., Drysdale R. B. The purification and properties of histidinol dehydrogenase from Neurospora crassa. Biochem J. 1967 Apr;103(1):36–41. doi: 10.1042/bj1030036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elmore D. T., Roberts D. V., Smyth J. J. Kinetics and mechanism of catalysis by proteolytic enzymes: The kinetics of hydrolysis of derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine(thialysine)by bovine trypsin. Biochem J. 1967 Mar;102(3):728–734. doi: 10.1042/bj1020728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elmore D. T., Smyth J. J. A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate. Biochem J. 1968 Mar;107(1):103–107. doi: 10.1042/bj1070103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson Q. H., Milnes L. Apparatus for rapid and sensitive spectrophotometry. Biochem J. 1964 Apr;91(1):161–171. doi: 10.1042/bj0910161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. HEIN G., NIEMANN C. An interpretation of the kinetic behavior of model substrates of alpha-chymotrypsin. Proc Natl Acad Sci U S A. 1961 Sep 15;47:1341–1355. doi: 10.1073/pnas.47.9.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaiser E. T., Awazu S., Carson F. W. The hydrolysis of O-hippuryglycolate catalyzed by carboxypeptidase A. Evidence for possible allosteric effects. Biochem Biophys Res Commun. 1965 Dec 9;21(5):444–447. doi: 10.1016/0006-291x(65)90402-x. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. SMITH E. L., PARKER M. J. Kinetics of papain action. III. Hydrolysis of benzoyl-L-arginine ethyl ester. J Biol Chem. 1958 Dec;233(6):1387–1391. [PubMed] [Google Scholar]
  13. TROWBRIDGE C. G., KREHBIEL A., LASKOWSKI M., Jr SUBSTRATE ACTIVATION OF TRYPSIN. Biochemistry. 1963 Jul-Aug;2:843–850. doi: 10.1021/bi00904a037. [DOI] [PubMed] [Google Scholar]

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