Fluorescence properties of neutral protease from Bacillus subtilis.
The fluorescence properties of neutral protease from B. subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977). In the pH range 5-9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm. At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shift emission, appear rather buried in the hydrophobic protein interior. Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues. On the other hand, removal of both zinc and calcium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium. Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants. Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60-70 degrees. Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents. The results reveal differences in the fluorescence properties of the tryptophanyl residues of B. subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins.
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D005453:Fluorescence; D006146:Guanidines; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis / Q000379:methods; D013820:Thermolysin / Q000032:analysis
D006146:Guanidines; D010447:Peptide Hydrolases; D013820:Thermolysin