Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1979 Nov 1;183(2):389–394. doi: 10.1042/bj1830389

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

G B Irvine, D T Elmore
PMCID: PMC1161570  PMID: 43135

Abstract

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.

Full text

PDF
390

Selected References

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

  1. Barrett A. J. Cathepsin D. Purification of isoenzymes from human and chicken liver. Biochem J. 1970 Apr;117(3):601–607. doi: 10.1042/bj1170601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayliss R. S., Knowles J. R., Wybrandt G. B. An aspartic acid residue at the active site of pepsin. The isolation and sequence of the heptapeptide. Biochem J. 1969 Jun;113(2):377–386. doi: 10.1042/bj1130377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Husain S. S., Ferguson J. B., Fruton J. S. Bifunctional inhibitors of pepsin. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2765–2768. doi: 10.1073/pnas.68.11.2765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Irvine G. B., Elmore D. T. The use of a radiochemical titrant for the determination of the operational molarity of solutions of pepsin. Biochem Soc Trans. 1976;4(4):643–645. doi: 10.1042/bst0040643. [DOI] [PubMed] [Google Scholar]
  5. Kazakova O. V., Orekhovich V. N., Pourchot L., Schuck J. M. Effect of cathepsins D from normal and malignant tissues on synthetic peptides. J Biol Chem. 1972 Jul 10;247(13):4224–4228. [PubMed] [Google Scholar]
  6. Keilová H., Bláha K., Keil B. Effect of steric factors on digestibility of peptides containing aromatic amino acids by cathepsin D and pepsin. Eur J Biochem. 1968 May;4(4):442–447. doi: 10.1111/j.1432-1033.1968.tb00232.x. [DOI] [PubMed] [Google Scholar]
  7. Keilová H. Inhibition of cathepsin D by diazoacetylnorleucine methyl ester. FEBS Lett. 1970 Feb 25;6(4):312–314. doi: 10.1016/0014-5793(70)80086-2. [DOI] [PubMed] [Google Scholar]
  8. Lundblad R. L., Stein W. H. On the reaction of diazoacetyl compounds with pepsin. J Biol Chem. 1969 Jan 10;244(1):154–160. [PubMed] [Google Scholar]
  9. Perlmann G. E. EFFECT OF SOLVENTS AND OF TEMPERATURE ON THE OPTICAL ROTATORY PROPERTIES OF PEPSIN. Proc Natl Acad Sci U S A. 1959 Jul;45(7):915–922. doi: 10.1073/pnas.45.7.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Rajagopalan T. G., Stein W. H., Moore S. The inactivation of pepsin by diazoacetylnorleucine methyl ester. J Biol Chem. 1966 Sep 25;241(18):4295–4297. [PubMed] [Google Scholar]
  11. Santi D. V., McHenry C. S., Perriard E. R. A filter assay for thymidylate synthetase using 5-fluoro-2'-deoxyuridylate as an active site titrant. Biochemistry. 1974 Jan 29;13(3):467–470. doi: 10.1021/bi00700a011. [DOI] [PubMed] [Google Scholar]
  12. Schnabel E. Verbesserte Synthese von tert.-Butyloxycarbonyl-aminosäuren durch pH-Stat-Reaktion. Justus Liebigs Ann Chem. 1967;702:188–196. doi: 10.1002/jlac.19677020123. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES