Watts B P, Barnard M, Turrens J F
Department of Biomedical Sciences, College of Allied Health Professions, University of South Alabama, Mobile 36688-0002.
Arch Biochem Biophys. 1995 Mar 10;317(2):324-30. doi: 10.1006/abbi.1995.1170.
Exposure of proteins to ONOO- (fatty acid-free bovine serum albumin (BSA) and histones, 10 mg/ml) was accompanied by light emission which could be detected using a photon counter. Light emission upon addition of ONOO- to either histones or BSA increased linearly with ONOO- concentration at a rate of 50 +/- 4 and 66 +/- 4 cps/(mg protein.mM ONOO-), respectively (averages+SE). Bicarbonate (25 mM) increased ONOO(-)-dependent BSA chemiluminescence approximately 3-fold above baseline (221 +/- 6 cps/(mg protein.mM ONOO-)). The peak of peroxynitrite-dependent light emission was around 40-fold higher than when 1 mM tert-butyl-hydroperoxide (t-BOOH) and 1.6 microM hemin were used as oxidants. Fatty acid-containing BSA (0.04-0.08%) emitted 3.4-fold more light than pure BSA. Chemiluminescence increased with pH, being 4.5-fold higher at pH 8.8 than at pH 6.0. However, the half-life of emissive species did not change with pH, suggesting that the process leading to the formation of electronically excited states is the same at all pHs. Tryptophan or N-acetyltyrosine oxidation by ONOO- was accompanied by chemiluminescence (130 +/- 10 and 14 +/- 3 cps/(mg amino acid.mM ONOO-), respectively). Exposure of DNA or isolated nucleotides to either t-BOOH/hemin or ONOO- was not accompanied by light emission. Leptomonas seymouri (an insect parasite used as a model of intact cells) exposed to ONOO- emitted 3700 +/- 400 cps/(mg protein.mM ONOO-), compared to 55 +/- 3 cps/(mg protein.mM peroxide) when t-BOOH was used as oxidant. While chemiluminescence of L. seymouri exposed to ONOO- increased measured at concentrations as low as 30 microM, carbonyl formation (from protein oxidation) and thiobarbituric acid-reactive substances (lipid peroxidation) could be measured only if cells were exposed to initial ONOO- larger than 700 microM. Spectral analysis suggests that excited carbonyls (emission wavelength 340-450 nm) are not produced in high proportions. A substantial amount of light is generated above 500 nm, part of which could come from triplet states of tryptophan and tyrosine.
蛋白质与过氧亚硝酸根（ONOO-）（无脂肪酸牛血清白蛋白（BSA）和组蛋白，10 mg/ml）接触时会伴随发光现象，可用光子计数器检测到。向组蛋白或BSA中添加ONOO-后产生的发光强度随ONOO-浓度呈线性增加，速率分别为50±4和66±4 cps/（mg蛋白质·mM ONOO-）（平均值±标准误）。碳酸氢盐（25 mM）使依赖于ONOO-的BSA化学发光比基线水平（221±6 cps/（mg蛋白质·mM ONOO-））增加约3倍。与使用1 mM叔丁基过氧化氢（t-BOOH）和1.6 μM血红素作为氧化剂时相比，过氧亚硝酸根依赖的发光峰值高出约40倍。含脂肪酸的BSA（0.04 - 0.08%）发出的光比纯BSA多3.4倍。化学发光随pH值升高而增加，在pH 8.8时比pH 6.0时高4.5倍。然而，发光物质的半衰期不随pH值变化，这表明在所有pH值下导致电子激发态形成的过程是相同的。ONOO-氧化色氨酸或N-乙酰酪氨酸时伴随化学发光（分别为130±10和14±3 cps/（mg氨基酸·mM ONOO-））。DNA或分离的核苷酸与t-BOOH/血红素或ONOO-接触时不会伴随发光现象。感染锥虫（一种用作完整细胞模型的昆虫寄生虫）与ONOO-接触时发出3700±400 cps/（mg蛋白质·mM ONOO-）的光，而以t-BOOH作为氧化剂时为55±3 cps/（mg蛋白质·mM过氧化物）。虽然感染锥虫与低至30 μM的ONOO-接触时化学发光就会增加，但只有当细胞接触初始浓度大于700 μM的ONOO-时才能检测到羰基形成（源于蛋白质氧化）和硫代巴比妥酸反应性物质（脂质过氧化）。光谱分析表明，激发态羰基（发射波长340 - 450 nm）产生的比例不高。在500 nm以上会产生大量光，其中一部分可能来自色氨酸和酪氨酸的三重态。
