Single-molecule reactions in liposomes
Single-molecule reactions in liposomes
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DOI：
10.1002/anie.200702348
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Yeung, Edward S.
中科院分区：
文献类型：
--
作者：
Hsin, Tse-Ming;Yeung, Edward S.
单分子研究可以揭示通常在批量实验中平均的随机行为。乳酸脱氢酶 (LDH)[1, 2] 和碱性磷酸酶 (ALP)[3, 4] 单分子活性的测量已有报道。当单个酶分子积累的荧光产物被限制在毛细管或微型小瓶中的 nL 至 pL 体积时，我们发现酶的单个分子表现出五倍 [1] 至 20 倍之间的活性。 [3]有几个不同的因素可以解释活动的广泛且非高斯分布。首先，蛋白质的糖基化 [3, 5] 影响其灵活性和动态稳定性。然而，所研究的 hLDH-H4 样品中不存在糖型和亚型。[1, 2] 此外，仅糖基化不能解释参考文献 [3] 中的活性分布，因为它已被证明仅导致反应性的四倍变化。 [5]其次，每个反应区中仅存在一种分子的证明仅基于统计分析和稀释因子。第三，不能排除壁效应的影响。在此，我们报告了在仅存在一个酶分子和不存在壁相互作用的条件下测定单分子酶活性的方法，这些条件都通过直接观察得到证实。为了模拟生命系统中的生化转化并避免壁效应，反应在脂质体中进行[6-11]，脂质体也用作超小型容器。包括电融合、[6-8]电注射、[9, 10]和光诱导融合[12]在内的显微操作已被用来启动脂质体中的反应。诸如染料嵌入 DNA、[8] 酶催化反应 [9, 10] 以及聚合物与金属离子形成水凝胶的反应 [13] 等反应已有报道。然而，之前的单脂质体实验尚未达到单分子域。 [9, 10] 在本研究中，TOTO-3标记的ALP被限制在一个脂质体中，而二磷酸荧光素（FDP）被限制在另一个脂质体中。通过电融合混合脂质体的内容物。酶促反应产生荧光素（F），
Single-molecule studies can reveal stochastic behavior that is normally averaged out in bulk experiments. Measurements of the single-molecule activities of lactate dehydrogenase (LDH)[1, 2] and of alkaline phosphatase (ALP)[3, 4] have been reported. When fluorescent products accumulated from individual enzyme molecules were confined in nL to pL volumes in capillaries or in microfabricated vials, it was found that individual molecules of the enzymes exhibit activities that varied between five-[1] to 20-fold.[3] Several different factors may account for the broad and non-Gaussian distribution of activities. First, glycosylation of proteins [3, 5] affects both their flexibility and dynamic stability. Glycoforms and isoforms, however, were not present in the hLDH-H4 samples that were studied.[1, 2] Also, glycosylation alone cannot account for the distribution of activities in reference [3], since it has been shown to cause only a fourfold variation in reactivity.[5] Second, proof of the existence of only one molecule in each reaction zone was based solely on statistical analysis and dilution factors. Third, contributions from wall effects cannot be excluded. Herein, we report a determination of single-molecule enzyme activities under conditions such that the presence of only one enzyme molecule and the absence of wall interactions were both confirmed by direct observation.To mimic biochemical transformations in living systems and to avoid wall effects, reactions were carried out in liposomes [6–11] that also served as ultrasmall containers. Micromanipulation including electrofusion,[6–8] electroinjection,[9, 10] and light-induced fusion [12] have been used to initiate reactions in liposomes. Reactions such as intercalation of dye into DNA,[8] catalytic reactions of enzymes,[9, 10] and reactions of polymers with metal ions to form hydrogels [13] have been reported. However, previous single-liposome experiments have yet to reach the single-molecule domain.[9, 10] In this study, TOTO-3-labeled ALP was confined in one liposome, and fluorescein diphosphate (FDP) was confined in another. The contents of the liposomes were mixed by electrofusion. The enzymatic reaction produced fluorescein (F), which was