DS-835: Short Column Sedimentation Equilibrium Analysis for Rapid Characterization of Macromolecules in Solution

Short column sedimentation equilibrium analysis offers many advantages for researchers wanting quick, sure characterization of the solution properties of macromolecules. Presented here is a list of when short column methods are an appropriate choice, a brief theoretical treatment and an overview of short column sedimentation methods, including ones diagnostic for self-association and nonideality. Short column methods were first described by Van Holde and Baldwin in 1958 (1), and centerpieces specifically designed for these methods were described by Yphantis in 1960 (2). Their distinguishing characteristics are (Fig. 1): 1) the parallel rows of four, small, sample viewing (1.2 mm diameter) holes 4 mm apart, 2) the two rows of larger (2.4 mm) holes used for filling the cell, and 3) the connecting groove between the fill hole and sample viewing holes. Despite their early development, short column sedimentation equilibrium methods did not enjoy extensive use with the early users of sedimentation equilibrium for several reasons: 1) manual data acquisition from short columns required nearly the same amount of time as from longer columns and did not yield as much information per data set; 2) they required higher concentrations of material; 3) they could not be used with the photoelectric scanner of the Model E; and 4) they often required that a separate experiment be conducted to measure the sample concentration. However, a great deal has changed in the intervening years, and all of these limitations have been overcome. Data acquisition takes seconds now, whereas it could require days in the early ’60s. Thus, there is a distinct advantage to the rapid equilibration available with short columns in that they provide at least a 20-fold increase in the number of samples that can be examined in a given period of time. Thus, with a few exceptions, the quantity of Thomas M. Laue University of New Hampshire Department of Biochemistry Durham, NH 03824