Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis

Abstract

A polyacrylamide-gel electrophoresis method was developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 .mu.g/gel) or a solution of double-stranded plasmid PED 822 DNA (20 .mu.g/gel), and electrophoresis began. At the end of the run the gels were stained and the effect of temperature on mobility was observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and pig heart lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (Staphylococcus aureus .beta.-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton (1979). The method also resolved a complex mixture of double stranded DNA restriction-digest fragments.