Structural and functional alterations of two multidomain oxidoreductases induced by guanidine hydrochloride.
The unfolding and refolding of two multidomain oxidoreductases, bovine liver catalase and flavoprotein bovine milk xanthine oxidase (XO), have been analyzed by fluorescence spectroscopy, circular dichroism, and activity measurements. Two intermediates, a partially folded active dimer disassembled from the native tetramer and a partially folded inactivated monomer, are found to exist in the conformational changes of catalase induced by guanidine hydrochloride (GdnHCl). Similarly, two intermediates, an active, compacted intermediate bound by flavin adenine dinucleotide (FAD) partially and an inactive flexible intermediate with FAD completely dissociated, exist in the conformational changes of XO induced by GdnHCl. The activity regains completely and an enhancement in activity compared with the native catalase or native XO is observed by dilution of catalase or XO incubated with GdnHCl at concentrations not >0.5 or 1.8 M into the refolding buffer, but the yield of reactivation for catalase or XO is zero when the concentration of GdnHCl is >1.5 or 3.0 M. The addition of FAD provides a remarkable protection against the inactivation of XO by GdnHCl under mild denaturing conditions, and the conformational change of XO is irreversible after FAD has been removed in the presence of a strong denaturing agent. These findings provide impetus for exploring the influences of cofactors such as FAD on the structure-function relationship of xanthine oxidoreductases.
Jiao
Ming
M
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Zhou
Yu-Ling
YL
Li
Hong-Tao
HT
Zhang
De-Ling
DL
Chen
Jie
J
Liang
Yi
Y
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism / Q000706:statistics & numerical data; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D010088:Oxidoreductases; D019791:Guanidine