Determination of binding site concentration and average binding constant by the method of equilibrium sorption: Evidence for lysine as a principal receptor site for collagen-enzyme binding

The contribution of lysyl ϵ-amino groups to the noncovalent binding on enzymes to collagen has been determined by the method of equilibrium sorption. The sorption or binding of enzyme by chemically modified, and by the corresponding unmodified membraneous collagen, was determined as a function of enzyme concentration. Chemical modification of lysyl ϵ-amino groups was accomplished by carbamylation of the collagen membrane with potassium cyanate. The collagen membranes were contacted with a purified β-galactosidase (E. coli K₁₂). The enzyme was purified by affinity chromato- graphy. Analysis of the resultant sorption isotherms allowed determination of the heterogeneity index (a) and the concentration of active binding sites (A_o). Binding constants (K_o) and the free energy of binding (δG) were also computed from the sorption data.The results of these studies have shown that lysyl ϵ-amino groups of collagen are primary receptor sites for enzyme binding (β-galactosidase, E. coli K₁₂) and that carbamylation of the collagen membrane resulted in a decrease in active binding sites with little or no change in the binding constant of the remaining sites.