James B. Ifft

The buoyant titration of bovine serum mercaptalbumin I. An interpretation in terms of ion binding

Abstract The buoyant density of bovine serum mercaptalbumin was measured in CsCl throughout the pH range 5.0–12.5. The buoyant densities were found to increase gradually until a sharp increase was noted at pH 10.5. These data were analyzed in terms of changes in water and salt binding as the ionizable amino acid residues titrate. The recent data of Foster were used to compute the charge on the molecule as a function of pH. A generalized equation expressing the buoyant density was combined with the condition that the molecule be electrically neutral to yield the number of Cs + and Cl − bound as a function of pH. The results indicate a generalized increase and decrease in the number of Cs + and Cl − , respectively, bound as the pH is raised from the isoelectric pH of 5.3 to pH 9.5. As the pH continues to rise, a large increase in the number of Cs + bound is noted as the tyrosines are ionized, and a sharp decrease in the number of Cl − bound as the lysine residues are deprotonated.

PROTEINS AT SEDIMENTATION EQUILIBRIUM IN DENSITY GRADIENTS

Publisher Summary This chapter focuses on proteins at sedimentation equilibrium in density gradients. The distribution of a solute through the centrifuge cell at sedimentation equilibrium can be obtained from either a thermodynamic or a kinetic point of view. As the former method requires almost no assumptions, and the nature of the variables such as density is not open to question, this derivation can be considered. The thermodynamic requirement for a system to be at equilibrium at every point in the phase is that the total potential be the same throughout the phase. The observations as to predicted densities of proteins in solution are gross approximations for a number of reasons. Proteins are formed by dehydration reactions between amino acids. Examination of amino acid densities alone neglects the less-dense water molecule that is lost and does not take into account the contribution of the peptide bond to the density.

Sedimentation Equilibrium of Proteins in Density Gradients

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.