Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence.

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and that tryptophyl residues moved to a localization near the active site upon activation.