Continuous optical scanning in polyacrylamide gel electrophoresis: Estimation of the apparent diffusion coefficient of β-lactoglobulin B

Abstract

The continuous scanning apparatus developed by Catsimpoolas was applied to an analysis of the concentration profiles of a protein, β-lactoglobulin B, while it was subjected to polyacrylamide gel electrophoresis (PAGE) in a multiphasic buffer system. Continuous optical scanning in PAGE permitted reliable estimation of the standard deviation of the concentration profile (σ), the relationship between σ 2 and time, and the apparent diffusion coefficient, D′, derived from σ 2, as the current density varied from 2 to 9 mA/cm 2, protein load varied from 250 to 900 μg/cm 2, and the ionic strength varied from 0.015 to 0.065 m. Under these conditions, D′ was linearly related to current density and protein load. Further, log ( D′) was linearly related to gel concentration (% T) ranging from 6 to 14%. However, D′ was nonlinearly related to ionic strength. Due primarily to the ionic strength factor, the apparent diffusion coefficient of protein in gels appeared to be approximately 10-fold larger than under the conditions of high ionic strength conventionally used in sedimentation and diffusion studies. Extrapolation of D′ to 0% T, zero protein load, zero current density, and “infinite” ionic strength (assuming noninteraction of these factors), as well as correction for viscosity and temperature, yielded an estimated free-diffusion coefficient, D 20,w, of 3.1 × 10 −7 cm 2/s, which is compatible with previously reported values. These studies indicate that the optimal resolution obtained by PAGE will be considerably lower than that predicted theoretically on the basis of free-diffusion coefficients, and suggest that electrostatic interaction between the proteins and/or deformation of voltage gradient and pH within the protein zones may contribute significantly to band spreading.