A fluorimetric study of the role of calcium ions in the stability of thermolysin

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Abstract
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 10 mM-EDTA, which removed the functional Zn2+ and the 4 Ca2+ molecule from the enzyme. At 25.degree. C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290 nm, was essentially characteristic of tryptophan, with an emission maximum at 333 nm. The emission maximum of the apoenzyme is red-shifted to 338 nm 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. Fluorescence quenching studies using KI, N''-methylnicotinamide HCl and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. Whereas the native enzyme is heat-stable up to about 80.degree. C, for the apoenzyme a thermal transition is observed near 48.degree. 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. The functional Zn2+ does not contribute noticeably to the stability of thermolysin. Apparently, a major role in the structural stability of thermolysin is played by the Ca2+, which have a bridging function within this disulfide-free protein molecule.