Quantitative urea gradient gel electrophoresis for studies of dissociation and unfolding of oligomeric proteins

Abstract

Urea gradient gel electrophoresis combined with quantitative image processing of stained gels was used to analyze the dissociation and unfolding of the catalytic subunit of aspartate transcarbamoylase. The subunit, composed of three identical polypeptide chains, dissociates reversibly at high urea concentrations into unfolded chains. A comparison of the complex, but reproducible, gel patterns obtained for the native subunit and for the denatured protein in 6 M urea revealed significant differences at intermediate urea concentrations due to the presence of a transient kinetic intermediate identified as a relatively compact monomer. Mass transport equations based on a three state model were used to describe the urea gradient gel electrophoresis experiments, and a numerical solution yielded estimates of the population of molecular species and kinetic constants for the unfolding and refolding reactions as well as the dissociation and reconstitution reactions.