Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation; Mass spectrometry analysis of oxidation products

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. Irradiation of butyrylcholinesterase (BChE) in the presence of methylene blue results in a variety of oxidations typical of phenothiazine photochemistry of proteins (hydroxylated methionine, hydroxylated tryptophan, kynurenine, N-formyl-kynurenine, hydroxylated and peroxidized-cysteine), but in addition there are novel oxidations (oxidation of serine, oxidation of histidine, 3-OH-kynurenine) and there is extensive protein degradation such that when the enzymatic activity is finally eliminated there is little to no intact protein remaining.