A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium.
A method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alterations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loading compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i.e., without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from 'sensitive' regions of the discriminator map.
Hsu
C S
CS
Section on Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20894.
Minton
A P
AP
D002499:Centrifugation, Density Gradient; D002918:Chymotrypsin / Q000737:chemistry; D046911:Macromolecular Substances; D008433:Mathematics; D008956:Models, Chemical; D012996:Solutions; D014361:Trypsin Inhibitors / Q000737:chemistry
D046911:Macromolecular Substances; D012996:Solutions; D014361:Trypsin Inhibitors; D002918:Chymotrypsin