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

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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. 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.

Original languageEnglish (US)
Pages (from-to)336-348
Number of pages13
JournalPhotochemistry and Photobiology
Volume89
Issue number2
DOIs
StatePublished - Mar 1 2013

Fingerprint

Butyrylcholinesterase
Methylene Blue
methylene blue
Mass spectrometry
Mass Spectrometry
mass spectroscopy
Kynurenine
Oxidation
oxidation
proteins
products
methionine
histidine
tryptophan
cysteine
irradiation
Irradiation
Histidine
Tryptophan
Methionine

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Biochemistry
  • Medicine(all)

Cite this

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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. 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.",
author = "Ozden Tacal and Bin Li and Oksana Lockridge and Schopfer, {Lawrence M}",
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T1 - Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation; Mass spectrometry analysis of oxidation products

AU - Tacal, Ozden

AU - Li, Bin

AU - Lockridge, Oksana

AU - Schopfer, Lawrence M

PY - 2013/3/1

Y1 - 2013/3/1

N2 - 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.

AB - 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.

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