Reactivity of the invariant cysteine of silver hake parvalbumin (Isoform B) with dithionitrobenzoate (DTNB) and the effect of differing buffer species on reactivity

Covalent and non-covalent homo- and hetero-dimerization has been implicated as a means of functional regulation in members of the EF-hand family of ca lcium-binding proteins, including oncomodulin, S100, troponin C and calmodu lin. The presence of an invariant cysteine at position 18 in all beta-linea ge parvalbumins suggests an important function for this sulfhydryl-containi ng amino acid. The chemical reactivity of Cys-18 toward the thiol-directed reagent dithionitrobenzoate (DTNB) (in a variety of solvent systems) was ex amined in this study to obtain information on differences in reactivity of the group in the calcium-bound versus the metal-free protein. Under our exp erimental conditions, the purified parvalbumin isoform from the silver hake employed was shown to be a mixture of monomeric protein (with a free thiol at the Cys-18 position) and disulfide-linked 'dimeric' protein. The monome ric protein, but not the disulfide-linked 'dimeric' protein, proved to be r eactive towards DTNB and it was determined that the reaction occurs at a si gnificantly greater rate with protein in the presence of excess EDTA than w ith just the calcium-loaded protein, indicating a conformational change in the ape-protein which exposes the Cys-18 thiol to the sulfhydryl-directed r eagent. This is in agreement with the observations of Clayshulte et al. ( 1 990) on oncomodulin, and also with Donato and Martin (1974) in their work w ith carp beta-parvalbumin, but in contrast to the observations of Mutus et al. (1988) also with oncomodulin. The experimental kinetic profiles associa ted with the reaction of DTNB and SHPV-B were fitted to a simple model cont aining multiple bi-molecular reactions. The results of molecular dynamics s imulations, focused on the issue of solvent exposure of the Cys-ls thiol, a re consistent with our interpretation of the kinetics. The reactivity of Cy s-18 with DTNB predictably increased with pH, but, quite unexpectedly, the precise nature of the kinetic response was also found to depend rather dram atically on the nature of the buffer species employed. The buffer dependenc y of the reaction of DTNB with the Cys-18 sulfhydryl group is one of the mo re significant, and unexpected, findings of this study. The dramatic change s in Cys-18 modification kinetic profiles observed with buffers of differen t ionic character (NH4HCO3, Tris-HCl, piperazine) clearly demonstrate that buffer selection is not a trivial matter. Buffers that have significant met al ion complexing power can easily sequester trace metal ions, and protein thiol-disulfide kinetic studies performed in the presence of such buffers c an exhibit dramatically altered kinetic behavior when compared to similar s tudies performed in metal-free buffer systems. (C) 1999 Elsevier Science In c. All rights reserved.