Microphotometric analysis of protein-bound thiols and disulfides with an azogenic maleimide.

Abstract

The chromogenic thiol reagent N-(4-aminophenyl)-maleimide (APM) was used to provide a sensitive procedure for the determination of protein-bound thiol group, both before and after reduction of disulfide bonds by tri-n-butylphosphine. The bound reagent is diazotized and coupled at low pH to N-(1-naphthyl)ethylenediamine to form an intense magenta label (lambda max 547 nm) with a high molar absorbancy (epsilon 547 = 49,800 cm2/mmol). The validity of absolute quantitation afforded by the method was shown by comparing the results with standard chemical analyses of thiol contents in a model system. Furthermore, the applicability of the procedure to biological objects was shown on isolated epididymal bull sperm heads.