Photosensitized oxidation of 2′,7′-dichlorofluorescin:: Singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′-dichlorofluorescein
Photosensitized oxidation of 2′,7′-dichlorofluorescin:: Singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′-dichlorofluorescein
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DOI：
10.1016/s0891-5849(02)00982-6
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发表时间：
2002-10-01
影响因子：
7.4
通讯作者：
Chignell, CF
中科院分区：
文献类型：
--
作者：
Bilski, P;Belanger, AG;Chignell, CF
2‘，7’-二氯荧光素(DCFH)常通过监测其高荧光氧化产物2‘，7’-二氯荧光素(DCF)的出现来评估细胞内的氧化应激。我们研究了DCFH在溶液中的光敏氧化反应，阐明了单分子氧(O-1(2))在该反应中所起的作用。我们使用了孟加拉(RB)、原卟啉和DCF作为光敏剂。在DCFH(50um)存在下，Rb(20um)在50 mM磷酸盐(pH 7.4)中照射(550 Nm)导致DCF的快速形成，测量结果是其特征吸光度和荧光增加。在脱氧溶液中氧化速度较快，在D2O中不增加，在叠氮化钠存在下甚至增加。抗氧化剂的存在与O-1(2)反应，从而去除氧气，加速DCF的形成。这些结果排除了O-1(2)在DCF形成中的任何潜在的直接参与，即使DCFH是一种有效的(物理)猝灭剂O-1(2)(k(Q)=1.4×10(8)M-1 S(-1)在甲醇中)。DCF也是O-1(2)的中等光敏剂，在D2O中的量子产率约为phi=0.06，在碳酸亚丙酯中的量子产率约为phi=0.08，这明确地表明DCF在紫外光和可见光激发下可以以三重态存在。这个三联体可以通过氧化还原和自由基机制(S)启动DCFH的光氧化，类似于涉及Rb的机制(见上文)。我们的结果表明，在光照下，DCF可以作为一种温和的光敏剂引发DCFH的氧化，这可能会引发和加速DCF的形成。我们还表明，虽然O-1(2)对DCF的产生没有直接贡献，但它可以通过与产生过氧化产物和过氧基的细胞底物反应间接地产生DCFH，这些产物和过氧基能够在随后的暗反应中氧化DCFH。这些发现表明，DCFH不应被视为对单重态分子氧敏感的探针，在使用DCFH测量由于可见光和紫外光暴露而导致的细胞氧化应激时必须谨慎。爱思唯尔科学公司出版。
2',7'-Dichlorofluoresein (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (O-1(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 muM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 muM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D2O, and even increased in the presence of sodium azide. The presence of antioxidants that react with O-1(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of O-1(2) in DCF formation, even though DCFH is an efficient (physical) quencher of O-1(2) (k(q) = 1.4 X 10(8) M-1 s(-1) in methanol). DCF is also a moderate photosensitizer of O-1(2) with a quantum yield of circa phi = 0.06 in D2O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while O-1(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings Suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure. Published by Elsevier Science, Inc.