Alarcón Emilio, Henríquez Carola, Aspée Alexis, Lissi Eduardo A
Facultad de Química, Departamento de Química Física, Pontificia Universidad Católica de Chile, Santiago, Chile.
Photochem Photobiol. 2007 May-Jun;83(3):475-80. doi: 10.1562/2006-07-25-RA-983.
Low level chemiluminescence (CL) is observed after protein oxidation mediated by singlet oxygen produced in Rose Bengal (RB) irradiation. This CL lasts for several minutes after the end of the photolysis. In this work, the mechanism of the process was assessed from the spectral characteristics of the CL and the effect of antioxidants (Trolox or ascorbate), Ebselen (a compound with peroxidase-like activity), azide (a singlet oxygen scavenger) and D2O, added prior to or after RB irradiation. It is concluded that most of the light emission is due to formation of excited states generated in the decomposition of peroxides and/or hydroperoxides accumulated during the photolysis. Experiments carried out in the presence of several amino acids (Cys, Met, His, Tyr and Trp) and di- and tripeptides suggest that peroxides (and/or hydroperoxides) of Trp residues are mainly responsible for the CL observed after singlet oxygen-mediated protein oxidation. The much weaker CL observed after the oxidation of proteins without Trp residues supports this conclusion. A comparison of the results obtained employing free Trp, Ala-Trp and Trp-Ala dipeptides, Ala-Trp-Ala tripeptide and Trp-containing proteins supports the conclusion that blocking the amino group of the Trp moiety strongly increases the efficiency of the chemiluminescent process, producing approximately 2.5x10(-8) photons per oxidized Trp group in Ala-Trp. A mechanism comprising two chemiluminescent oxidation pathways of Trp residues is proposed to explain the results.
在孟加拉玫瑰红（RB）照射产生的单线态氧介导的蛋白质氧化后，可观察到低水平化学发光（CL）。这种CL在光解结束后持续几分钟。在这项工作中，从CL的光谱特征以及抗氧化剂（生育三烯酚或抗坏血酸盐）、依布硒仑（一种具有过氧化物酶样活性的化合物）、叠氮化物（一种单线态氧清除剂）和重水（D2O）在RB照射之前或之后添加的效果评估了该过程的机制。得出的结论是，大部分发光是由于在光解过程中积累的过氧化物和/或氢过氧化物分解时产生的激发态的形成。在几种氨基酸（半胱氨酸、蛋氨酸、组氨酸、酪氨酸和色氨酸）以及二肽和三肽存在下进行的实验表明，色氨酸残基的过氧化物（和/或氢过氧化物）是单线态氧介导的蛋白质氧化后观察到的CL的主要原因。在没有色氨酸残基的蛋白质氧化后观察到的弱得多的CL支持了这一结论。对使用游离色氨酸、丙氨酰 - 色氨酸和色氨酰 - 丙氨酸二肽、丙氨酰 - 色氨酰 - 丙氨酸三肽以及含色氨酸蛋白质获得的结果进行比较，支持了这样的结论，即封闭色氨酸部分的氨基会大大提高化学发光过程的效率，在丙氨酰 - 色氨酸中每个氧化的色氨酸基团产生约2.5×10⁻⁸个光子。提出了一个包含色氨酸残基的两条化学发光氧化途径的机制来解释这些结果。
