Evidence for the Existence of Cross-Linked Intermediates during Unfolding and Refolding of CK in UGGE

Urea gradient gel electrophoresis (UGGE) is an important technique for studying the conformation changes of proteins during denaturation. This paper reports on an investigation of the unfolding and refolding of creatine kinase (CK) by UGGE. The native and denatured CK underwent electrophoresis in polyacrylamide gels containing a linear 0-8 mol/L gradient of urea perpendicular to the direction of migration. The results showed that unfolding and refolding of CK is a relatively rapid process. The denatured enzyme could refold to a conformation with activity during electrophoresis at low urea concentrations, indicating that denaturation in urea is reversible. More importantly, both the native and denatured CK were separated into multiple parallel bands through UGGE, but the bands decreased significantly when mercaptoethanol was added to the samples. The results suggest that various kinds of unfolding and refolding intermediates were formed during UGGE, which are assumed to be oligomers with disulfide bonds between peptide chains. Urea/SDS (sodium dodecylsulphate) polyacrylamide two-dimensional electrophoresis proved that these unfolding and refolding intermediates formed during UGGE were oligomers which were composed of different number of subunits cross-linked by disulfide bonds. The results indicate that the unfolding and refolding of CK are relatively rapid processes with some cross-linked intermediates with disulfide bonds during unfolding and refolding of the enzyme.

Keywords
creatine kinase; unfolding and refolding; urea gradient gel electrophoresis; intermediate; disulfide bonds