QUENCHING OF ROOM-TEMPERATURE PROTEIN PHOSPHORESCENCE BY ADDED SMALL MOLECULES
QUENCHING OF ROOM-TEMPERATURE PROTEIN PHOSPHORESCENCE BY ADDED SMALL MOLECULES
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DOI：
10.1021/bi00422a026
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发表时间：
1988-11-01
期刊：
影响因子：
2.9
通讯作者：
VANDERKOOI, JM
中科院分区：
文献类型：
--
作者：
CALHOUN, DB;ENGLANDER, SW;VANDERKOOI, JM
确定了一些可以有效地猝灭色氨酸室温磷光的分子试剂，并检查了它们猝灭9种蛋白质中色氨酸磷光寿命的能力。对于所有猝灭剂，猝灭效率通常遵循相同的顺序，即，N-乙酰基天冬酰胺（NATA）>小清蛋白。乳球蛋白核糖核酸酶T1 >肝醇脱氢酶>醛缩酶>链霉蛋白酶。麻仁蛋白>天青蛋白>碱性磷酸酶。猝灭速率常数为O2和CO是相对不敏感的蛋白质的差异，而H2S和CS2有点更敏感。这些小分子试剂似乎通过渗透到蛋白质中起作用。然而，渗透到真正掩埋的Dahans是不太有利的比以前建议的;在五个蛋白质研究，O2的淬灭效率是20-1000倍低于NATA，高达105低H2S和CS2。更大和更极性的淬灭剂。包括有机硫醇、共轭酮和酰胺以及阴离子物质。并进行了比较。这些猝灭剂的效率与猝灭剂的大小或极性无关，猝灭反应具有低活化能，并且猝灭速率对溶剂粘度不敏感。这些结果表明，较大的猝灭剂不接近掩埋的色氨酸通过渗透到蛋白质中，即使在长的磷光时间尺度，也是不一致的机制，其中猝灭剂遇到色氨酸发生在自由溶液中，如在蛋白质开放反应。所获得的结果表明，淬火过程涉及一个长距离的无辐射转移。此外，埋葬度和保护淬火之间的强烈的明显相关性是一致的，预期的远距离转移机制的急剧距离依赖性。由于共振能量（福斯特）转移的条件不存在，淬灭反应似乎可能通过激发三重态中的掩埋色氨酸与溶液中附近的淬灭剂之间的电子转移或交换而发生。
A number of molecular agents that can efficiently quench the room temperature phosphrescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequences, namely, N-acetyltryptophanamide (NATA) > parvalbumin .apprxeq. lactoglobulin .apprxeq. ribonuclease T1 > liver alcohol dehydrogenase > aldolase > Pronase .apprxeq. edestin > azurin > alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 105 lower for H2S and CS2. Larger and more polar quenchers .sbd. including organic thiols, conjugated ketones and amides, and anionic species .sbd. were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer. In addition, the strong apparent correlation between degreee of burial and protection against quenching is consistent with the sharp distance dependence expected for a long-range transfer mechanism. Since the conditions for resonance energy (Forster) transfer do not exist, it seems probable that the quenching reaction occurs by way of an electron transfer or exchange between the buried tryptophan in the excited triplet state and the quencher nearby in solution.