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

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Abstract
Hemocyanin of the blue crab C. sapidus has the typical structure of crustacean hemocyanins in that its smallest in vivo structure is a hexamer of subunits each having a MW of .apprx. 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 O2 binds to the binuclear Cu site has been reported to be very sensitive to pH and to a variety of inorganic allosteric effectors. The interaction of L-lactate, a natural metabolite, with the native hemocyanin and with chromatographically purified hexamers and dodecamers is reported. Under ionic conditions that approximate those found physiologically, the addition of 10 mM L-lactate to native Callinectes hemocyanin substantially increases its O2 affinity (.DELTA. log P50 = -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 O2 binding sites. Independent measurements by ultrafiltration techniques indicated a dissociation constant of 3.2 mM with 2.8 lactate binding sites per 6 O2 binding sites. The 2 sets of data clearly indicate that there is < 1 lactate binding site per O2 binding site. The fit to the titration was not improved with the assumption of > 1 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 5 major bands. Two of these bands, which constitute 1/6 of the total dodecameric hemocyanin, do not appear on gel electrophoresis of dissociated hexamers. The O2 affinities of the hexameric and dodecameric hemocyanin forms are similar to one another but show differences in their sensitivity to L-lactate. The O2 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. The magnitude of the Bohr effect was unchanged by the addition of L-lactate over the pH range 7.5-8.0. There is no significant effect of L-lactate on the aggregation state of the hemocyanin or on its 340-nm Cu-O2 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 oxyhemocyanin.