Determination and Derivatization of Protein Thiols by n-Octyldithionitrobenzoic Acid

Abstract

n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman′s reagent, can be isolated in crystalline form. Like Ellman′s reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman′s reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman′s reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.