Tyrosine fluorescence as a measure of denaturation in thermolysin

The heat and guanidine hydrochloride denaturation of thermolysin has been followed by fluorescence techniques. The native enzyme has a single emission peak which is decreased in intensity and which splits into two clearly resolved peaks upon denaturation. These data are interpreted to indicate that energy transfer from tyrosine to tryptophan occurs in the native enzyme which is lost upon denaturation. Even though zinc is fully bound to thermolysin at 90 degrees C or in the presence of 6 M guanidine hydrochloride, removal of zinc from the denatured enzyme has no effect on the emission spectrum. Removal of Ca2+ from the denatured enzyme. These data indicate that even though the metal ions are bound to the denatured protein, they provide little structural integrity to the protein as measured by energy transfer between tyrosine and tryptophan.