Kinetic Studies of the Reaction of Thiol Groups of Calf‐Thymus Histone F3 with 5‐5′‐Dithiobis(2‐nitrobenzoic acid)

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Abstract
The reactivity of 5‐5′‐dithiobis(2‐nitrobenzoic acid) with thiol groups of calf thymus histone F3 was studied by using the tripeptide glutathione and 2‐mercaptoethanol as models. The results found for glutathione suggest that the reaction is dependent on the nucleophile‐S− concentration. The reactivity of thiol groups of the histone in 2.4 mM sodium phosphate solutions at room temperature was found to be much higher than for glutathione and almost independent of pH between pH 5 to 7. The reaction follows second order kinetics, K2(app.) at pH 5 being 4000 times higher than K2 for glutathione. The pKa(app.) for thiol groups of histone F3 was estimated to be 4.2 indicating the predominance at low pH of ionic forms of the thiol groups. At 24 mM sodium phosphate, pH 6.6, 80% of thiol groups react at a similar high rate as in the case of 2.4 mM sodium phosphate, and the remaining 20% reacts very slowly, the kinetics being of first order. Local denaturation is not obtained by urea treatment and high temperatures (85°C), but guanidine hydrochloride completely unfolds the microenvironments containing cysteine. It is proposed that such reacting microenvironments are important in the biological role of the histone F3.