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Biophys J, October 2001, p. 1868-1880, Vol. 81, No. 4

Non-Ideality by Sedimentation Velocity of Halophilic Malate Dehydrogenase in Complex Solvents

Alexandra Solovyova,*dagger Peter Schuck,Dagger Lionel Costenaro,* and Christine Ebel*

 *Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale J. P. Ebel, F-38027 Grenoble, France;  dagger Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, 61015 Kharkov, Ukraine; and  Dagger Division of Bioengineering and Physical Science, Office of Research Services, National Institutes of Health, Bethesda, Maryland 20892 USA

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D° (1 + kDc), and the reciprocal sedimentation coefficient s = s°/(1 + ksc). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for ks and s° and also for D° and the buoyant molar mass were obtained. It was also possible to obtain good estimates for kD and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Biophys J, October 2001, p. 1868-1880, Vol. 81, No. 4
© 2001 by the Biophysical Society   0006-3495/01/10/1868/13  $2.00


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