title: 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 1.8-1.4M urea) and I(2) (existing in 0.8-0.4M 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.
authors: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. qguo@sibs.ac.cn
authors: Guo
authors: G
authors: Qin
authors: Zhang
authors: Z
authors: Jianwei
authors: Liu
authors: L
authors: Taotao
authors: Wang
authors: W
authors: Xicheng
mesh_terms: D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D047528:Stichopus / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology*
chemical_list: D004220:Disulfides; D014508:Urea; D001122:Arginine Kinase