DIFFERENCES IN THE CHEMICAL-REACTIVITY OF INDIVIDUAL MOLECULES OF AN ENZYME
DIFFERENCES IN THE CHEMICAL-REACTIVITY OF INDIVIDUAL MOLECULES OF AN ENZYME
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DOI：
10.1038/373681a0
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发表时间：
1995-02-23
期刊：
影响因子：
64.8
通讯作者：
YEUNG, ES
中科院分区：
文献类型：
--
作者：
XUE, QF;YEUNG, ES
最近，许多注意力集中在单个分子的检测和物理表征上（1-11）。使用这种方法来研究化学性质，如反应性，单分子提供了研究这些如何从分子到分子变化的潜力，并且对于作为时间函数的单个分子，生物分子如酶的复杂结构使它们成为研究分子水平上的细微变化或差异如何影响化学反应性的特别有吸引力的目标。我们之前已经证明（12，13），可以使用荧光微量测定法研究非常少量（zeptomole）的酶;通过荧光显微镜也可以在油分散液滴中检测到单个酶分子（14，15）。本文观察了乳酸脱氢酶（LDH-1）分子与烟酰胺腺嘌呤二核苷酸（NAD（+））反应，当它们在很低的浓度下存在于狭窄的毛细管中时，每个酶分子产生一个离散的NADH区;这些可以通过荧光光谱学进行操作和监测，我们发现，单个纯酶分子的活性可以变化高达四倍，并且这些活性在两小时内保持不变。我们认为，活性差异的起源可能在于存在几种稳定形式的酶。
MUCH attention has been focused recently on the detection and physical characterization of individual molecules(1-11). Using such methods to study the chemical properties, such as reactivity, of single molecules offers the potential to investigate how these might vary from molecule to molecule, and for individual molecules as a function of time, The complex structures of biomolecules such as enzymes make them particularly attractive targets for studying how subtle changes or differences at the molecular level might influence chemical reactivity. We have shown previously(12,13) that very small (zeptomole) amounts of enzymes can be studied using a fluorescence microassay; single enzyme molecules have also been detected in oil-dispersed droplets by fluorescence microscopy(14,15). Here we report the observation of reactions of individual molecules of lactate dehydrogenase (LDH-1), which produces NADH from lactate and nicotinamide adenine dinucleotide (NAD(+)), When they are present at very low concentrations in a narrow capillary, each enzyme molecule produces a discrete zone of NADH; these can be manipulated electrophoretically and monitored by fluorescence spectroscopy, We find that the activity of individual electrophoretically pure enzyme molecules can vary by up to a factor of four, and that these activities remain unchanged over a two-hour period. We suggest that the origin of the activity differences may lie in the presence of several stable forms of the enzyme.