Renaturation studies of free and immobilized D-amino-acid oxidase.
The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.
D000818:Animals; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011489:Protein Denaturation; D013552:Swine
D004800:Enzymes, Immobilized; D006146:Guanidines; D003605:D-Amino-Acid Oxidase; D019791:Guanidine