1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn2+ ion and the four Ca2+ ions/molecule from the enzyme. 2. At 25°C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N′-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80°C, for the apoenzyme a thermal transition is observed near 48°C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn2+ ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca2+ ions, which have a bridging function within this disulphide-free protein molecule.
The role of calcium ions in the stability and instability of a thermolysin-like protease
