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Preferential interaction measurements between proteins and monosodium glutamate were carried out to arrive at an understanding of the mechanism of its strong effect on tubulin stability and self-assembly into microtubules. For all proteins studied, i.e. bovine serum albumin, lysozyme, beta-lactoglobulin, and calf brain tubulin, the protein showed a large preferential hydration in the presence of monosodium glutamate. The enhancement of tubulin self-association by monosodium glutamate can be interpreted in terms of the large unfavorable free energy of interaction between the additive and the protein. Preferential interactions were also examined for lysine hydrochloride, which also gave a preferential hydration of the proteins, except for tubulin. The dependence of the preferential hydration parameter on proteins was different for the two additives, suggesting the importance of net electrostatic charges of proteins in their interaction with glutamate anions and lysinium cations. The zero preferential interaction of lysine hydrochloride with tubulin indicates an affinity of the lysine cation for the protein. Both additives increased the transition temperature of proteins. This can be understood in terms of the unfavorable free energy of interaction between the additive and the protein surface, which should be even more unfavorable when the denaturation causes an increase in the surface area.

The interaction of the antimitotic drug vincristine with tubulin has been investigated by the techniques of self-assembly, velocity sedimentation, fluorescence, circular dichroism, and differential spectroscopy. Vincristine has been shown to inhibit the self-assembly of tubulin into microtubules at substoichiometric concentrations. The sedimentation velocity patterns at low vincristine concentration (less than 1 X 10(-5) M to 7 X 10(-5) M) consist of a bimodal boundary with a 5.8 S peak and a fast moving peak, with a nominal S20,w value of 9 S. The data conform to the ligand-promoted self-association theory of Cann and Goad (Cann, J.R., and Goad, W.B. (1972) Arch. Biochem. Biophys. 153, 603-609). At higher vincristine concentrations (greater than 8 X 10(-5 M), most of the protein is polymerized and sediments as a hypersharp peak with a nominal S20,w value of approximately 20 S. The association constant for the binding of vincristine to tubulin, determined by spectrofluorometry, is 3.5 X 10(4) liters/mol at 25 degrees C. The binding of vincristine does not induce any significant conformational changes in tubulin; however, the difference spectral results indicate perturbation of both vincristine and protein chromophores.