Monitoring the reactions of single enzyme molecules and single metal ions
Monitoring the reactions of single enzyme molecules and single metal ions
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DOI：
10.1021/ac970631k
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发表时间：
1997-10-15
影响因子：
7.4
通讯作者：
Yeung, ES
中科院分区：
文献类型：
--
作者：
Tan, WH;Yeung, ES
研究单分子的一种方法是通过重复的反应来循环分子，即监测感兴趣的分子催化的反应的产物。通过使用基于激光的光学显微镜和CCD检测系统，我们已经确定了单个酶分子的化学活性。单个分子被捕获在飞升大小的小瓶(膜上的孔或通过光刻制造的纳米二氧化硅小瓶)中，直径从5到12个CRM，深4到6微米，作为单独的纳米反应器，通过在这些反应器中填充极低浓度的乳酸脱氢酶(LDH-1)、过量乳酸和烟酰胺腺嘌呤二核苷酸(NAD(+))来分离单个乳酸脱氢酶(LDH-1)分子。可以同时实时跟踪100多个独立反应堆中的荧光产物NADH。观察到不同的反应特征，包括瞬时反应速率和30分钟间隔内的平均反应速率。我们发现，即使在微环境相同的情况下，单个酶分子的活性也表现出异质性，这表明在其他相同的分子中可能存在不同的分子构象。我们进一步将这一技术用于单金属离子催化反应的研究，观察到对单个Os(VIII)离子的催化活性是均匀的。
One approach for studying single molecules is to cycle the molecule through a reaction repeatedly, i.e., to monitor the products from a reaction catalyzed by the molecule of interest. By using a laser-based optical microscope and a CCD detection system, we have determined the chemical activities of individual enzyme molecules. Single molecules are trapped inside femtoliter-size vials (pores in membranes or nanoscopic silica vials manufactured by photolithography) ranging from 5 to 12 Crm in diameter and from 4 to 6 mu m deep, which act as individual nanoscopic reactors, Single lactate dehydrogenase (LDH-1) molecules are isolated by filling these reactors with very low concentrations of LDH-1, excess lactate, and nicotin-amide adenine dinucleotide (NAD(+)). The fluorescent product NADH in more than 100 individual reactors can be followed simultaneously in real time. Different reaction characteristics are observed, both in terms of the instantaneous reaction rates and the average rates over a 30-min interval. We found that even when the microenvironments are identical, individual enzyme molecules reveal heterogeneities in their activities, This indicates that distinct molecular conformations may exist in otherwise identical molecules. We have further used this technique for the study of single metal ion-catalyzed reactions, Uniform catalytic activities are observed for individual Os(VIII) ions.