The gel-filtration behaviour of proteins related to their molecular weights over a wide range

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Abstract
Correlation between elution volume, Ve, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 ([alpha]-crystallin) on Sephadex G-200 columns at pH 7.5. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free golbular proteins fitted a smooth Ve-log (mol. wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. Ve-log (mol. wt.) curves based on results with the three gels are taken to represent the behaviour of "typical" globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. Some glycoproteins, including [gamma]- globulins and fibrinogen, do not conform to the standard relationship. The effects of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually > 106. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250 000. The free sub-units readily lose enzymic activity. Recognition of the atypical gel-filtration behaviour of [gamma] -globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128 000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140 000; milk alkaline phosphatase, 180 000.