A special role of phosphate in the stability of lactate dehydrogenase against destruction by a polyelectrolyte

It has been shown by X-ray structure analysis that proteins have specific anion-binding sites for sulfate, citrate, and phosphate ions; however, the functional role of these anions is not always clear. Thus, it is unknown which of two phosphate anions, mono- or divalent, determines the stability of cellular proteins under stress conditions. In the present work, the influence of phosphate, sulfate, and chloride on the stability of lactate dehydrogenase (LDH) in the presence of poly(styrenesulfonate) (PSS) has been investigated by the methods of steady-state kinetics and intrinsic protein fluorescence. The study is based on the analysis of differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of the concentrations of mono- and bivalent phosphate forms differs, whereas sulfate remains in the bivalent form. It was shown that the differences between the influence of phosphate and sulfate ions at pH 7.0 were greater; divalent phosphate ions much more effectively stabilized LDH against destruction by a polyelectrolyte compared with sulfate and monovalent phosphate. It was concluded that, of two anion-binding sites of the LDH molecule, the intersubunit center plays the most important role in its stabilization against destruction by polyelectrolyte, and, of two forms of phosphate anions, its bivalent form HPO ₄ ^?2 plays the stabilizing role.