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

Biochemistry. 1978 Nov 14;17(23):5000-5. doi: 10.1021/bi00616a022.

Abstract

The binding of Lens culinaris lectin to the receptors of rabbit erythrocytes was studied by a quantitative fluorometric method using the fluoresceinated conjugate. Equilibrium data showed the presence of 1.2 x 10(6) receptors/cell with an association constant varying from 8 to 3 x 10(6) M-1 over a temperature range of 5 to 37 degrees C. The binding reaction is exothermic with a deltaH = -4.6 kcal/mol and a deltaS = + 15 eu. It appears that most of the bound lectin molecules had only a single site occupied, the agglutination being due to a very low fraction (0.0003--0.0056) of lectin molecules with two 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 x 10(2) M-1 was calculated for methyl alpha-d-glucoside and 9 x 10(2) for methyl alpha-D-mannoside. The association and dissociation rate constants of the binding reaction are, respectively, in the range of 3--10 x 103 and 3--33 x 10(-4) M-1 s-1 in the temperature range of 5 to 37 degrees 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 measurements.

MeSH terms

  • Animals
  • Erythrocytes / metabolism*
  • Kinetics
  • Lectins*
  • Mathematics
  • Protein Binding
  • Rabbits
  • Spectrometry, Fluorescence
  • Thermodynamics

Substances

  • Lectins