ELECTROPHORETIC ANALYSIS OF THE UNFOLDING OF PROTEINS BY UREA

The unfolding of several proteins by urea has been followed by electrophoresis of a band of protein through a slab gel of polyacrylamide in which there was a gradient of urea concentration perpendicular to the direction of electrophoresis. Unfolding was invariably manifested by a marked reduction of mobility, presumably due to molecular sieving of the expanded polypeptide chain by the polyacrylamide gel. The procedure provides a continuous two-dimensional pattern of the effect of urea on the shape of the protein and is especially sensitive to microheterogeneity of the protein. Experiments with pancreatic trypsin inhibitor, ribonuclease, lysozyme, chymotrypsin, chymotrypsinogen, staphylococcal nuclease, and cytochrome c were consistent with the results of others using orthodox methods and confirm the validity of the method. Where unfolding occurred, it was generally rapidly reversible and the curves were entirely consistent with the presence of only the native and the fully unfolded states. Serum albumin gave more complex curves and a remarkable illustration of micro-heterogeneity. β-Lactoglobulins A and B and ovalbumin refold very slowly and the unfolded molecules appeared to equilibrate preferentially with compact, but non-native, forms at low urea concentrations. © 1979.