Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.