Abstract
The neutral protease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His228 of the former one, indicating that His228 was photooxidized. This result suggests that His228 is involved in the catalytic reaction of the neutral protease or interaction with substrates.
