Equilibrium and kinetic studies of the binding of Lens culinaris lectin to rabbit erythrocytes by a quantitative fluorometric method

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Abstract
The binding of L. culinaris lectin to the receptors of rabbit erythrocytes was studied by a quantitative fluorometric method using the fluoresceinated conjugate. Equilibrium data showed 1.2 .times. 106 receptors/cell with an association constant varying from 8-3 .times. 106 M-1 over a temperature range of 5-37.degree. C. The binding reaction was exothermic with a .DELTA.H [enthalpy] = -4.6 kcal/mol and a .DELTA.S [entropy] = +15 eu [entropy units]. Most of the bound lectin molecules apparently had only a single site occupied, the agglutination being due to a very low fraction (0.0003-0.0056) of lectin molecules with 2 sites occupied. The specificity of the reaction was demonstrated through its inhibition by nonfluoresceinated LL and by the specific sugars methyl .alpha.-D-glucoside and methyl .alpha.-D-mannoside. From the inhibition curves an association constant of 4 .times. 102 M-1 was calculated for methyl .alpha.-D-glucoside and 9 .times. 102 for methyl .alpha.-D-mannoside. The association and dissociation rate constants of the binding reaction are, respectively, in the range of 3-10 .times. 103 and 3-33 .times. 10-4 M-1 s-1 in the temperature range of 5-37.degree. C. The activation energies of the forward and reverse reactions are 7 and 13 kcal/mol, respectively. The association constants and the binding enthalpy calculated from the activation energies were fully consistent with those obtained by equilibrium measurement.