PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN
PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN
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DOI：
10.1111/j.1751-1097.1973.tb06392.x
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发表时间：
1973-01-01
影响因子：
3.3
通讯作者：
GROSSWEINER, LI
中科院分区：
文献类型：
--
作者：
KEPKA, AG;GROSSWEINER, LI
研究表明，在曙红对蛋清溶菌酶的光动力灭活中，单线态氧是主要的氧化实体，使用D2O来延长溶剂诱导的衰变寿命，叠氮离子作为一种特定的清除剂。根据敏化剂对酶是游离的还是络合的，可以区分两种失活状态。基于光静态测量和失活量子产率的自由染料敏化动力学分析表明，至少有1/15的单线态氧与溶菌酶相互作用导致溶菌酶的裂解活性丧失。三重态曙红的直接攻击在空气饱和溶液中的总体贡献较小，在空气饱和溶液中，四分之一的反应会导致失活。溶菌酶在pH 4-12范围内结合1个曙红分子，导致色氨酸残基荧光几乎完全猝灭而不抑制酶活性。灭活量子产率表明，结合染料产生的单线态氧是钝化剂，但主要攻击发生在溶菌酶分子的络合部分。尽管量子产率增加了5-10倍，但从H2O到D2O的色氨酸残基损失相同或更小，这表明单线态氧也通过与色氨酸以外的残基反应而失活。光化学和荧光结果与色氨酸108位点与曙红结合位相一致，也是单线态氧的反应靶点。在对早期关于曙红敏化光氧化I“的工作的重新审查中，已经发现单线态氧是好氧溶液中的氧化剂。
It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo‐dynamic inactivation of hen egg white lysozyme by eosin, using D2O to enhance the solvent‐induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air‐saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H2O to D2O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re‐examination of earlier work on eosin‐sensitized photo‐oxidation of I“, it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.