Desacetamidocolchicine (DAAC) is a colchicine analogue that lacks the acetamido side chain in the ring-B of colchicine. Unlike colchicine, it binds to tubulin very quickly, and yet it has powerful antimitotic properties. It has been demonstrated that the beta-tubulin isoforms differ significantly in their interactions with colchicine. In an effort to understand the role of the ring-B of colchicine, we have studied the interaction of DAAC with purified beta-tubulin isoforms. The association was studied fluorometrically using a stopped-flow instrument under pseudo-first-order conditions in the presence of a large excess of drug. The observed pseudo-first-order rate constants increased in a nonlinear way with the drug concentration, indicating that the binding of DAAC to tubulin isoforms occur in two steps as is true for the binding of colchicine to tubulin (Garland, D.L. (1978) Biochemistry 17, 4266-4272), [formula: see text] where the first step is a fast reversible binding and the second step is a slow conformational change leading to the formation of the stable complex (T.DAAC)*. Kinetic analysis shows that the tubulin isoforms exhibit very little differences in their K1 values, which are 5794 +/- 670, 7109 +/- 1800, and 8993 +/- 1780 M-1 for alpha beta II, alpha beta III, and alpha beta IV, respectively. The k2 values for alpha beta II, alpha beta III, and alpha beta IV are 0.67 +/- 0.05, 0.05 +/- 0.006, and 0.59 +/- 0.07 s-1, respectively. The apparent on-rate constants (k(on,app) = K1k2) for alpha beta II, alpha beta III, and alpha beta IV are 3907 +/- 530, 376 +/- 80, and 5305 +/- 1200 M-1 s-1, respectively. The off-rate constants as measured by the loss of fluorescence of drug-tubulin complexes in the presence of a large excess of podophyllotoxin are 6.3 x 10(-4), 5.2 x 10(-4), and 5.7 x 10(-4) s-1, respectively, for alpha beta II, alpha beta III, and alpha beta IV. The affinity constants as determined by Scatchard analyses are 2.5 x 10(6), 1.5 x 10(6), and 4 x 10(6) M-1 for alpha beta II, alpha beta III, and alpha beta IV, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)