Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate

Hemocyanin of the blue crab Callinectes sapidus has the typical structure of crustacean hemocyanins in that its smallest in vivo structure is a hexamer of subunits each having a molecular mass of approximately 75 000. As found in the blood, Callinectes hemocyanin consists of a mixture of hexamers and dodecamers (typically 1:4). As in other crustacean hemocyanins, the affinity with which oxygen binds to the binuclear copper site has been reported to be very sensitive to pH and to a variety of inorganic allosteric effectors. We report here the interaction of L-lactate, a natural metabolite,with the native hemocyanin and with chromatographically purified hexamers and dodecamers. Under ionic conditions that approximate those found physiologically, the addition of 10 mM L-lactate to native Callinectes hemocyanin substantially increases its oxygen affinity (Δ log P(50) = -0.28). The data from lactate titrations were fit to a theoretical equation,and the best fit was obtained with a lactate dissociation constant of 1.8 mM for the oxy state and 2.2 lactate binding sites for every 6 oxygen binding sites. Independent measurements by ultrafiltration techniques indicated a dissociation constant of 3.2 mM with 2.8 lactate binding sites per 6 oxygen binding sites. The two sets of data clearly indicate that there is less than one lactate binding site per oxygen binding site. The fit to the titration was not improved with the assumption of more than one class of lactate binding site. The hexamers and dodecamers of native Callinectes hemocyanin are not in equilibrium and are stable after separation by gel-filtration chromatography. Polyacrylamide gel electrophoresis of the subunits of the dissociated dodecamers shows five major bands.Two of these bands, which constitute one-sixth of the total dodecameric hemocyanin, do not appear upon gel electrophoresis of dissociated hexamers. The oxygen affinities of the hexameric and dodecameric hemocyanin forms are similar to one another but show differences in their sensitivity to L-lactate.The oxygen affinity of native Callinectes hemocyanin was increased appreciably more by L-lactate than by glycolate,D-lactate, and pyruvate (listed in decreasing order of effectiveness).Propionate, acetate, succinate, D-alanine, and L-alanine were without effect, thus illustrating the selectivity of the L-lactate effect on the hemocyanin. We found the magnitude of the Bohr effect to be unchanged by the addition of L-lactate over the pH range 7.5-8.0. Moreover, there is no significant effect of L-lactate on the aggregation state of the hemocyanin or on its 340-nm copper-oxygen absorption band.The foregoing results are consistent with the role of L-lactate as a specific allosteric effector of Callinectes hemocyanin that acts by preferential binding to a stereospecific site of the oxy hemocyanin.