CHROMOPHORE-ASSISTED LASER INACTIVATION OF PROTEINS IS MEDIATED BY THE PHOTOGENERATION OF FREE-RADICALS
CHROMOPHORE-ASSISTED LASER INACTIVATION OF PROTEINS IS MEDIATED BY THE PHOTOGENERATION OF FREE-RADICALS
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DOI：
10.1073/pnas.91.7.2659
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发表时间：
1994-03-29
影响因子：
11.1
通讯作者：
JAY, DG
中科院分区：
文献类型：
--
作者：
LIAO, JC;ROIDER, J;JAY, DG
发色团辅助激光失活（CALI）是一种选择性地使感兴趣的蛋白质失活以阐明其体内功能的技术。该方法可应用于一系列广泛的生物学问题，并且为了合理应用它，需要了解其机制。我们在此报告，CALI不是由光诱导的热变性介导的，而是由光生自由基介导的。热扩散计算表明，CALI引起的温度变化太小，不足以导致热变性，并且CALI的阿伦尼乌斯曲线与光热机制不一致。CALI在阈值以上显示出能量剂量的倒数关系，并且可被自由基猝灭剂抑制，从而证明了蛋白质失活的光化学机制。对抑制CALI有效的猝灭剂类型表明，活性物质是一种氢提取剂，它不是来自分子氧。我们认为活性自由基物质是羟基自由基，其极短的寿命解释了CALI的空间特异性，即距离染料部分15埃内产生半数最大损伤，在34埃处没有明显损伤。这些数据与顺序双光子过程产生的自由基形成是一致的。
Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 angstrom from the dye moiety and no significant damage occurs at 34 angstrom. The data are consistent with free-radical formation resulting from a sequential two-photon process.