title: Photooxidation of histidine and tryptophan residues of papain in the presence of methylene blue.
abstract: Papain [EC 3.4.22.2] was photooxidized using methylene blue as a sensitizer. The photooxidzed enzyme lost its caseinolytic activity and had significantly decreased histidine and tryptophan contents. The tyrosine content was the same before and after the photooxidation. The SH content of the photooxidized enzyme, as determined after reduction with dithiothreitol, was also unchanged. The loss of histidine was always slower than the loss of enzymatic activity, being less than one residue per molecule even when the enzymatic activity was completely lost. However, the inactivation and the oxidation of a histidine residue were pH-dependent in a similar fashion in the pH range of 5.0-8.0, the pH profiles conforming to theoretical titration curves with apparent pKa values of 6.6 and 6.7, respectively. The fact that the ionization of a histidine residue in papain has a normal imidazole pKa value is entirely in accord with the finding for stem bromelain [EC 3.4.22.4] (Murachi, T., Tsudzuki, T., & Okumura, K. (1975) Biochemistry 14, 249-255), and is of great significance in relation to the mechanism of catalysis by these enzymes.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D004229:Dithiothreitol; D006639:Histidine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008751:Methylene Blue; D010084:Oxidation-Reduction; D010206:Papain / Q000378:metabolism; D010777:Photochemistry; D013438:Sulfhydryl Compounds / Q000032:analysis; D014364:Tryptophan / Q000032:analysis
chemical_list: D000596:Amino Acids; D013438:Sulfhydryl Compounds; D006639:Histidine; D014364:Tryptophan; D010206:Papain; D008751:Methylene Blue; D004229:Dithiothreitol