Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

doi:10.1042/BJ2150287

Paper

Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

Published Nov 1, 1983 · R. Cook, J. Powers

The Biochemical journal

Q1 SJR score

7

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0

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Abstract

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

Study Snapshot

Benzyl p-guanidinothiobenzoate hydrochloride is a new, flexible, and stable titrant for determining active-site concentrations of trypsin and related enzymes, offering advantages over p-nitrophenyl p'-guanidinobenz

PopulationOlder adults (50-71 years)

Sample size24

MethodsObservational

OutcomesBody Mass Index projections

ResultsSocial networks mitigate obesity in older groups.

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Paper

Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

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References

Citations

Bauhinia Proteinase Inhibitor-Based Synthetic Fluorogenic Substrates for Enzymes Isolated from Insect Midgut and Caterpillar Bristles

Bauhinia proteinase inhibitor-based synthetic substrates can be used to study trypsin-like enzyme activities in non-mammalian enzymes, offering an alternative to traditional methods.

Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents.

The best inhibitors for thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride, and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H

4-Acetamidophenyl esters and 4-acetamidoanilides of L-arginine,p-guanidino-L-phenylalanine, L-lysine, N2-[D-fructos-3-O-yl and D-glucos-3-O-yl]acetyl-L-lysine as potential acrosin inhibitors

These compounds show potential as weak inhibitors of human acrosin and bovine trypsin, but require further evaluation for their full potential.

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates.

Human beta-factor XIIa is the most reactive and substrate specific blood coagulation protease, with a kinetic constant range of over 470-fold.

Peptide thioester substrates for serine peptidases and metalloendopeptidases.

Peptide thioester substrates are sensitive and useful for studying enzyme specificity and inhibitors, especially in cases where other synthetic substrates are unavailable.