A fluorimetric study of the role of calcium ions in the stability of thermolysin.
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 Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees 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 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees 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 Zn(2+) 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 Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.
D002118:Calcium; D004492:Edetic Acid; D006146:Guanidines; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013820:Thermolysin; D014508:Urea
D006146:Guanidines; D014508:Urea; D004492:Edetic Acid; D013820:Thermolysin; D002118:Calcium