Intermediates in the inactivation and unfolding of dimeric arginine kinase induced by GdnHCl.
Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.
Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084 China.
Qin
Q
Guo
Feng
F
Zhao
Zhi
Z
Guo
Xicheng
X
Wang
D001122:Arginine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D011489:Protein Denaturation / Q000502:physiology; D013056:Spectrophotometry, Ultraviolet
D001122:Arginine Kinase; D019791:Guanidine