Electrostatic and conformational effects on the reaction of thiol groups of calf thymus histone H3 with 5,5′-dithiobis(2-nitrobenzoic acid)

The presence of highly basic proteins (histones or protamines), causes an increase in the rate of the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs2) with the tripeptide model glutathione. This effect is explained by considering that polycationic molecules, such as histones or protamines, can attract the negatively charged reacting molecules, thus producing a catalytic effect. This effect disappears at high ionic strength due to a shielding of the charges; Urea causes a shift to the K2(app) vs. pH curve for the histone H3-Nbs2 reaction. This shift (2.1 units of pH for 8 m urea) indicates that urea denatures, at least to some extent, the tertiary structure of the microenvironments containing cysteine of histone H3, but it is unable to eliminate an unspecific electrostatic effect (similar to that caused by polycations in the GSH-Nbs2 reaction), which also contributes to the increase of the reaction rate. Combined effects of urea and ionic strength on the reaction of GSH and of histone H3 with Nbs2 gives rise to shifts of both curves of K2(app) us. pH, approaching one to the other very closely. This is interpreted as due to the appearance of shielding effects on the electrostatic charges of the histone, and also of the small molecules. The greater efficiency of guanidine hydrochloride, compared to that of urea, in causing a shift of the rate constant curve of histone H3 is interpreted as due to a combined effect of denaturation and electrostatic shielding in the case of guanidine hydrochloride. © 1978.