QUANTITATION OF NONIDEAL BEHAVIOR IN PROTEIN SIZE-EXCLUSION CHROMATOGRAPHY

The size-exclusion chromatographic partition coefficient (K(SEC)) was measured on a Superose 6 column for three sets of well-characterized spherically symmetrical solutes: the compact, densely branched non-ionic polysaccharide, Ficoll; the flexible chain non-ionic polysaccharide, pullulan; and compact, anionic synthetic polymers, carboxylated starburst dendrimers. All three solutes display a congruent dependence of K(SEC) on solute radius, R. In accord with a simple geometric model for SEC, all of these data conform to the same linear plot of K(SEC)1/2 vs. R. This plot reveals the behavior of non-interacting spheres on this column. Comparison of results for a number of globular proteins at various pH values to this ''ideal'' curve allows a quantitative measure of protein attraction or repulsion. It is shown that the usual procedure of obtaining a ''best-fit'' curve for a set of proteins is likely to generate an erroneous calibration curve.