Use of Trinitrophenylation for Quantification of Protease and Peptidase Activities

V. I. Surovtsev^*, T. V. Fjodorov, A. N. Baydus, M. A. Borozdina, V. V. Gusev, and V. P. Chuprunov

State Research Center for Applied Microbiology, Ministry of Health of the Russian Federation, Obolensk, Moscow Region, 142279 Russia; fax: (0967) 36-0010; E-mail: center@gncpm.serpukhov.su

^* To whom correspondence should be addressed.

Received July 26, 2000; Revision received October 31, 2000

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A sensitive and precise method for quantifying protease and peptidase activities is suggested. N-Terminal amino groups of peptides which are formed during hydrolysis of the substrates react with trinitrobenzenesulfonic acid (TNBS), and the trinitrophenyl (TNP) derivatives are determined spectrophotometrically. Spontaneous hydrolysis of TNBS is considerably diminished on trinitrophenylation at pH 7.4 rather than at pH 9-10 as is usually used. The trinitrophenylation method can be used to determine the initial rate of hydrolysis and the kinetics of reactions catalyzed by proteases and peptidases.

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KEY WORDS: trinitrophenylation, protease, peptidase, amino group, modification

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