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.
Guo
Qin
Q
Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084 China.
Zhao
Feng
F
Guo
Zhi
Z
Wang
Xicheng
X
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