Binding of colchicine to beef brain tubulin: influence of methodology on binding constants

We have evaluated the effects that methodology (incubation time, concentration of beef brain tubulin) have on the measurement of the interaction of tubulin with colchicine. The results show that, with DEAE filter paper assay, binding is maximal for 3 hr of incubation at 37 degrees C, and for low concentrations of tubulin (0.05 mg/ml). In these conditions binding constants determined with SCATCHARD plot are: constant of dissociation (KD) of complex tubulin-colchicine equal to 9.8 X 10(-7) M and stechiometry equal to 0.57 mole of colchicine bound for 1 mole of tubulin dimer. Competition of drugs with colchicine binding site may be evaluated with this methodology. IC50 and affinity constants (Ki) of podophyllotoxin, a competitive inhibitor of colchicine-binding determined with these experimental conditions were 1.3 X 10(-6) M and 1.1 X 10(-6) M, respectively.