Fluorescent thiol reagents. X. Fluorimetric estimation of the reactivity of thiols; examples in small molecular compounds, Taka-amylase A and myosin A.

Abstract

Abstract The kinetic analysis on the reactivity of organic thiols was carried out using a maleimide derivative, N-(p-(2-benzimidazolyl)phenyl)maleimide, by means of a novel fluorimetric determination of the simple linear equations giving the dissociation constant of a thiol, pKa, and the pH-independent second-order rate constant, κo. 1. 1. Kinetic parameters, pKa and κo, for seven organic thiols including aromatic compounds were fluorimetrically determined. The values obtained were in good agreement with those reported using different methods. 2. 2. The Bronstead relation on the data obtained was found to be fairly linear except thiosalicylic acid, which has a strong intramolecular hydrogen bond. 3. 3. pKa and κo of thiophenol simultaneously shshowed a biphasic response to the increasing polarity of solvents, while the value of pKa for thiosalicylic acid increased with decreasing polarity, without an appreciable increase in κo. 4. 4. The parameters for the single thiol per mole of Taka-amylase A were on the Bronstead plot in spite of the very low pKa value, 7.9–8.0. In contrast, those for myosin A possessing S1 modified sith N-ethylmaleimide were far from this straight line, the rate being markedly low relative to the pKa values. Substrates affected the state of the thiols for the both enzymes, but in different ways. The above results, obtained with a very small amount (usually, few mg) of a protein sample, have revealed that the proposed fluorimetric determination of the reaction rate of thiolsis simple and easy, although N-(p-(2-benzimidazolyl)phenyl)maleimide is somewhat labile in alkaline pH. The Bronstead relation thus obtained is effective for searching the state of thiol in macromolecules.