A nonlinear relationship between polyacrylamide gel electrophoresis (PAGE) retardation coefficients (KR) and molecular weights has been observed during analysis of several multimeric proteins. Although the deviation from linearity over a wide range of molecular weights is slight, it can lead to significant errors in the estimation of the sizes of individual multimers. Two alternative methods ofanalysis of PAGE results are compared and demonstrated to yield linear relationships for multimeric proteins having molecular weights as high as 900,000.
Polyacrylamide
gel electrophoresis (PAGE) has proven to be an extremely sensitive technique for the characterization of small amounts of protein and multiple forms of enzymes. Physical relationships among isozymes can be rapidly and conveniently examined using the procedures previously described (1,2). This entails the use of "Ferguson plots" (3) to discriminate among charge isomers, molecular weight isomers, and forms which differ with respect to both charge and size, as well as to estimate molecular weights. However, certain technical difficulties may be encountered when several multimers of a protein are to be analyzed. For example, the range of gel concentrations over which satisfactory results for all forms can be obtained may be restricted. Large multimers may exhibit minimum mobility even at moderate gel concentrations. Reduced resolution can also occur at low gel concentrations due to increased diffusion, particularly when aqueous mixtures are employed to detect the activity of enzymes in situ after PAGE.
Attempts to analyze the several forms of maize homoserine dehydrogenase (4) led to the present examination of PAGE results with multimeric proteins. It has been demonstrated that accurate Ferguson plots can be obtained by using a narrow range of gel concentrations and that such plots yield the intersecting pattern characteristic of molecular weight isomers. Although protein molecular weights between 45,000 and 500,000 are considered to be linearly related to the slopes of Ferguson plots (1,5). the present results suggest that this relationship is not strictly linear when multimeric proteins are examined. Two alternative treatments of PAGE results are shown to yield satisfactory linear relationships: one developed 513 Copyright