title: Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation; mass spectrometry analysis of oxidation products.
abstract: Methylene blue, 3, 7-bis(dimethylamino)-phenothiazin-5-ium chloride, is a reversible inhibitor of human butyrylcholinesterase (BChE) in the absence of light. In the presence of light and oxygen, methylene blue promotes irreversible inhibition of human BChE as a function of time, requiring 3 h irradiation to inhibit 95% activity. Inactivation was accompanied by a progressive loss of Coomassie-stained protein bands on native and denaturing polyacrylamide gels, suggesting backbone fragmentation. Aggregation was not detected. MALDI-TOF/TOF mass spectrometry identified oxidized tryptophan (W52, 56, 231, 376, 412, 490, 522), oxidized methionine (M81, 144, 302, 532, 554, 555), oxidized histidine (H214), oxidized proline (P230), oxidized cysteine (C519) and oxidized serine (S215). A 20 min irradiation in the presence of methylene blue resulted in 17% loss of BChE activity, suggesting that BChE is relatively resistant to methylene blue-catalyzed photoinactivation and that therefore this process could be used to sterilize BChE preparations.
authors.affiliation: Department of Biochemistry, School of Pharmacy, Hacettepe University, Ankara, Turkey.
authors.forename: Ozden
authors.initials: O
authors.lastname: Tacal
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Schopfer
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D002091:Butyrylcholinesterase / Q000737:chemistry*; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D006801:Humans; D008027:Light; D008751:Methylene Blue / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D055668:Photochemical Processes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D000596:Amino Acids; D002800:Cholinesterase Inhibitors; D002091:Butyrylcholinesterase; D010100:Oxygen; D008751:Methylene Blue