Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

Abstract

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I 1 (existing in 1.8–1.4 M urea) and I 2 (existing in 0.8–0.4 M urea). I 1 was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I 2 was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates ( I B). Comparison of the properties of these intermediates suggested that I B in the kinetic process corresponded to I 1 in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.