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Abstract

The two isothiocyanate groups of the anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2-2'-disulfonate (H₂DIDS) may react covalently with two lysine residues called a and b that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction leads to the establishment of intramolecular cross-links between a and b (M.L. Jennings & H. Passow, 1979, Biochim. Biophys. Acta 554:498-519). In the present work, the time course of the reactions with a and b, and of the establishment of the cross-link were investigated experimentally and compared with simple mathematical models of the reaction sequence. The rates of reaction with a and b were found to increase with increasing pH. Regardless of pH, the rate of reaction with a exceeds that with b several-fold. Once the H₂DIDS molecule has reacted with a, the rate of the subsequent reaction of the other isothiocyanate group with b is reduced by about 1/30. The reactions that follow the unilateral attachment to site b are not yet clear. A more detailed analysis of the time course of the cross-linking reaction suggests that a satisfactory description of the kinetics requires the assumption that the H₂DIDS binding site may exist in two different states, and that the transition from one state to the other is associated with changes of the reactivities of either lys a alone or of both lys a and b. This led to the formulation of the two-states model of the H₂DIDS binding site, which is supported by other pieces of independent evidence. The analysis of the pH dependence of the rate of thiocyanylation of b shows that the apparent pK value of that lysine residue is about 9.9 to 10.0 and hence slightly lower than the intrinsic pK of a lysine residue in an aqueous environment.