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

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: I1 (existing in 1.8–1.4 M urea) and I2 (existing in 0.8–0.4 M urea). I1 was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I2 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 (IB). Comparison of the properties of these intermediates suggested that IB in the kinetic process corresponded to I1 in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.