Synthesis of fluorescent organic phosphates and their equilibrium binding to bovine oxyhemoglobin

Fluorescent organic phosphates, beta-naphthyl diphosphate, beta-naphthyl triphosphate, and beta-naphthyl tetraphosphate, were synthesized from beta-naphthyl monophosphate using Pi and N,N'-dicyclohexylcarbodiimide. These organic phosphates were interacted with bovine oxyhemoglobin, all in no buffer, 0.1 M NaCl, at 25 degrees and in the pH range 5.5-7.0. Equilibrium binding parameters were determined by measuring the fluorescence quenching upon their interaction. It is indicated that bovine oxyhemoglobin has more than one binding site, one of which is very strong. The strength of binding to the stronger site is in the order beta-naphthyl tetraphosphate greater than beta-naphthyl triphosphate greater than beta-naphthyl diphosphate. The logarithms of association constants of these phosphates depend linearly on the net charges of these phosphates at any pH. The results were accounted for by electrostatic effects using a simple charge model. In that model, the average positive net charges in oxyhemoglobin involved in the binding of beta-naphthyl phosphate are shown as a function of pH. It is shown that the binding of these fluorescent organic phosphates is prevented reversibly by the excess addition of nonfluorescent organic and iorganic phosphates, inositol hexaphosphate, tripolyphosphate, and pyrophosphate. Assuming competitive binding in a single strong site, the association constants of these nonfluorescent phosphates were also determined by measuring the recovery of the fluorescence intensity upon the release of fluorescent phosphates. At pH 6.18, the association constant of pyrophosphate is lower than that of tripolyphosphate by one order.