Structural and functional alterations of two multidomain oxidoreductases induced by guanidine hydrochloride

The unfolding and refolding of two multidomain oxido- reductases, bovine liver catalase and flavoprotein bovine milk xanthine oxidase （XO）, have been analyzed by fluor- escence 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 gua- nidine hydrochloride （GdnHCI）. Similarly, two intermedi- ates, an active, compacted intermediate bound by flavin adenine dinucleotide （FAD） partially and an inactive flex- ible intermediate with FAD completely dissociated, exist in the conformational changes of XO induced by GdnHCl. The activity regains completely and an enhance- ment in activity compared with the native catalase or native XO is observed by dilution of catalase or XO incu- bated with GdnHCI 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 remark- able protection against the inactivation of XO by GdnHCi 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 find- ings provide impetus for exploring the influences of cofac- tors such as FAD on the structure-function relationship of xanthine oxidoreductases.