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Stabilization of Tubulin by Deuterium Oxide
Biochemistry (1999)
  • Gopal Chakrabarti, University of Kansas
  • Shane Kim, University of Kansas
  • Mohan L. Gupta, Jr., University of Kansas
  • Janice S. Barton, Washburn University
  • Richard H. Himes, University of Kansas
Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules
rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 °C. In H2O based
buffer, tubulin was completely inactivated after 40 h at 4 °C, but in buffer prepared in D2O, no
activity was lost after 54 h. Tubulin was completely inactivated at 37 °C in 8 h in H2O buffer, but only
20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower
rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both
temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer;
however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic
sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed
somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve
the disruption of hydrophobic forces. The conformational change is followed by an aggregation process
that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys.
Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also
observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon
structures and incompletely folded microtubules. When these polymers were disassembled and reassembled
in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO,
both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of
ribbon structures rather than microtubules.
Publication Date
Publisher Statement
Reprinted (adapted) with permission from Stabilization of Tubulin by Deuterium Oxide, Gopal Chakrabarti,, Shane Kim, Mohan L. Gupta, Jr., Janice S. Barton, and Richard H. Himes, Biochemistry 1999 38 (10), 3067-3072, DOI: 10.1021/bi982461r . Copyright 1999 American Chemical Society.
Citation Information
Gopal Chakrabarti, Shane Kim, Mohan L. Gupta, Janice S. Barton, et al.. "Stabilization of Tubulin by Deuterium Oxide" Biochemistry Vol. 38 Iss. 10 (1999) p. 3067 - 3072
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