Biochemistry zyxwvu 1981, 20, zyxwvu 5999-6005 5999 Cercek, L., Cercek, B., zyxwvutsrq & Ockey, C. H. (1978) Biophys. J. 23, 395-405. Costa, J. L., & Murphy, D. L. (1975) Nature (London) 255, 407-408. Crawford, N., Amos, L. A., & Castle, A. G. (1980) in Pla- telets - Cellular Response Mechanisms and Their Bio- logical Significance (Rotman, A., Meyer, F. A., Gitler, C., & Silberberg, A., Eds.) pp 171-188, Wiley, Chichester. Day, H. J., & Holmsen, H. (1971) Semin. Hematol.,4, 3-27. Ganesan, A. K., & Rotman, B. (1964) J. Mol. Biol. 10, Gordon, J. L., & Milner, A. J. (1976) in Platelets in Biology and Pathology (Gordon, J. L., Ed.) pp 3-23, North-Holland Publishing Co., Amsterdam. Inbar, M., Shinitzky, M., & Sachs, L. (1973) J. Mol. Biol. Lindmo, T., & Steen, H. B. (1977) Biophys. J. 18, 173-187. Nathan, I., Fleischer, G., Livne, A., Dvilansky, A., & Parola, 337-340. 81, 245-250. A. (1979) J. diol. Chem. 254, 9822-9828. Peerschke, E. zyxwv I., Zucker, M. B., Grant, R. A., Johnson, M. J., & Egan, J. J. (1980) Blood 55, 841-847. Perrin, E. (1926) J. Phys. Radium 7, 390-401. Pribluda, V., Laub, F., & Rotman, A. (1981) Eur. J. Biochem. Rotman, B., & Papermaster, B. W. (1966) Proc. Natl. Acad. Rotman, A., & Heldman, J. (1981) FEBSLett. 122,215-219. Shattil, S. J., & Copper, R. A. (1976) Biochemistry 15, Shinitzky, M., & Barenholz, Y. (1974) J. Biol. Chem. 249, Shinitzk), M., & Inbar, M. (1974) J. Mol. Biol. 85,603-615. Shinitzky, M., & Barenholz, Y. (1978) Biochim. Biophys. Udenfriend, S., Zaltzman-Nirenberg, P., & Guroff, G. (1966) White, J. G. (1971) in The Circulating Platelet (Johnson, S. 11 6, 293-296. Sci. U.S.A. 55, 134-141. 48 32-48 37. 2652-2657. Acta 515, 367-394. Arch. Biochem. Biophys. 116,261-270. A., Ed.) pp 45-121, Academic Press, New York. Changes in the Circular Dichroic Spectrum of Colchicine Associated with Its Binding to Tubulin+ H. William Detrich, 111, Robley C. Williams, Jr., Timothy L. Macdonald, Leslie Wilson, and David Puett* ABSTRACT: Circular dichroism has been used to study the interaction of colchicine with the tubulin zyxwvut a@ dimer at 26 OC. Tubulin purified from bovifie brain microtubule protein ex- hibits negligible circular dichroism at wavelengths above 3 10 nm. Free colchicine exhibits a negative circular dichroic band at 340 nm characterized by an extremum in molar ellipticity [e] of -3.35 (f0.27) zyxwvutsrq X 104 deg.cm*/dmol. This negative band either vanished or was greatly reduced, i.e., [e] = 0 (f0.57) X lo4 deg.cm*/dmol, when colchicine was bound to tubulin. This was demonstrated by circular dichroic studies on stable [ 3H]colchicine-tubulin complexes which were separated from all unbound colchicine by means of gel filtration. Also, tubulin was titrated with colchicine, and at low colchicine concen- trations, the observed ellipticity at 340 nm could be extrapo- Colchicine, the active antimitotic agent of the meadow saffron Colchicum autumnale, binds specifically and with high affinity to the tubulin dimer, the major protein subunit of ~~ 'From the Department of Biological Sciences (H.W.D. and L.W.), University of California, Santa Barbara, California 93 106, and the Departments of Molecular Biology (R.C.W.), Chemistry (T.L.M.),and Biochemistry (D.P.), Vanderbilt University, Nashville, Tennessee 37235 (R.C.W. and T.L.M.) and 37232 (D.P.). Received March 4, 1981; revised manuscript received June 22, 1981. A portion of this work was presented at the Second International Symposium on Microtubules and Microtubule Inhibitors, Aug 26-29, 1980, Beerse, Belgium, and the American Chemical Society Second Chemical Congress of the North American Continent, Aug 24-29, 1980, Las Vegas, NV. This research was supported by the National Institutes of Health (Research Grants NS13560, GM25638, and AM15838; Center Grant HD05797 provided necessary instrumentation) and the Vanderbilt Research Council. H.W.D. was a fellow in Cancer Research supported by Grant DRG 310-F of the Damon Runyon-Walter Winchell Cancer Fund and is currently supported by Grant GM07182 of the National Institutes of Health. T.L.M. is a Research Fellow of the Alfred P. Sloan Foundation (1981-1983), and D.P. was a Research Career Development Awardee during the course of these studies (AM00055). lated to zero at 0 M colchicine; saturation ~ u r r e d at a molar ratio of colchicine to tubulin of about 1:l. The association constant characterizing the drug-protein interaction was es- timated to be about 0.9 pM-l. As controls, three other acidic proteins were studied at a molar ratio of colchicine to protein of 2: 1, and their presence had no effect on the circular dichroic properties of colchicine. These results are consistent with the idea that a conformational change in colchicine accompanies its binding to tubulin. The spectrum of th? complex between 250 and 300 nm was quite similar to that expected from simple additivity of the spectra of drug and protein except between 255 and 265 nm. The technique describdd herein should be applicable to other protein-drug systems. microtubules (Shelanski & Taylor, 1968; Weisenberg et al., 1968; Wilson, 1970; Wilson & Meza, 1973). Approximately 1 mol of colchicine binds per mol of tubulin (Owellen et al., 1972; Wilson et al., 1974; Bhattacharyya & Wolff, 1976) with an affinity constant (for vertebrate tubulins) that ranges from 1 to 40 pM-', depending upon the source of tubulin and the conditions of measurement (Owellen et al., 1972,1974; Wilson et al., 1974; Bhattacharyya & Wolff, 1974, 1976; Sherline et al., 1975; McClure & Paulson, 1977). Colchicine binding requires native tubulin. Binding activity is abolished by de- naturants (Wilson, 1970) or by extensive treatment of tubulin with sulfhydryl reagents (Kuriyama & S a k i , 1974; Ikeda & Steiner, 1978). The colchicine binding activity of tubulin is also unstable in the buffers routinely used, decayihg according to first-order kinetics (Weisenberg et al., 1968; Wilson, 1970; Wilson & Meza, 1973; Sherline et ai., 1975; Cortese et al., 1977). The rate of decay is a sensitive function of pH and temperature, with optimal stability of binding activity observed at pH 6.75 and 0 OC (Wilson, 1970). Furthermore, the colchicine binding activity of tubClin is stabilized by vin- 0006-2960/8 1 /0420-5999$01.25/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1981 American Chemical Society