Nitrosylation of manganese(II) tetrakis(N-ethylpyridinium-2-yl)porphyrin: a simple and sensitive spectrophotometric assay for nitric oxide.
Reaction between NO(*) and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) was investigated at 25 degrees C. At high excess of NO(*) (1.5 mM) the reaction with the oxidized, air-stable form Mn(III)TE-2-PyP(5+) (5 microM), proceeds very slowly (t(1/2) congruent with 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, Mn(II)TE-2-PyP(4+), which reacts with NO(*) stoichiometrically and in the time of mixing (k congruent with 1 x 10(6) M(-1) s(-1)). The high rate of formation and the stability of the product, Mn(II)TE-2-PyP(NO)(4+) (¿Mn(NO)¿(6)), make the reaction outcompete the reaction of NO(*) with O(2). Our in vitro measurements show a linear absorbance response upon addition of NO to a PBS, pH 7.4, solution containing an excess of ascorbate over Mn(III)TE-2-PyP(5+). Thus, the observed interactions can be the basis of a convenient and sensitive spectrophotometric assay for NO(*). Also, it may have important implications for the in vivo behavior of Mn(III)TE-2-PyP(5+) which is currently exploited as a possible therapeutic agent for various oxygen-radical related disorders.
Spasojevic
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Batinic-Haberle
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Fridovich
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D001205:Ascorbic Acid / Q000378:metabolism; D002138:Calibration; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D009569:Nitric Oxide / Q000032:analysis* / Q000737:chemistry; D009603:Nitroso Compounds / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*
D008665:Metalloporphyrins; D009603:Nitroso Compounds; D009569:Nitric Oxide; C050950:tetrakis(N-methyl-4-pyridiniumyl)porphine manganese(III) complex; D001205:Ascorbic Acid; D010100:Oxygen