8 - Anal. Chem. 1997, 69, 4242-4248 Monitoring the...

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Monitoring the Reactions of Single Enzyme Molecules and Single Metal Ions Weihong Tan ² and Edward S. Yeung* Ames Laboratory s USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 One approach for studying single molecules is to cycle the molecule through a reaction repeatedly, i.e., to moni- tor 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 mol- ecules are trapped inside femtoliter-size vials (pores in membranes or nanoscopic silica vials manufactured by photolithography) ranging from 5 to 12 μ m in diameter and from 4 to 6 μ 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 instan- taneous reaction rates and the average rates over a 30- min interval. We found that even when the microenvi- ronments are identical, individual enzyme molecules reveal heterogeneities in their activities. This indicates that distinct molecular conformations may exist in other- wise 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. Studying single molecules can reveal heterogeneities among them. A variety of methodologies have been developed for single- molecule detection (SMD) and characterization. 1 - 10 In all these techniques, the amplification of the signal has been an important issue. The signals used in SMD are either optical or electro- chemical. Recent advances in optical detection, such as the avalanche photodiode and the intensified charge-coupled device (CCD), have enabled the efficient detection of single photons. Thus, SMD based on optical detection has become fairly straight- forward. Optical signal amplification is mainly performed through two different approaches. The first one is the direct detection of the photons produced by one molecule. It is, however, necessary to overcome photon-counting statistics. Even though single photons can be counted by a photon detector, many photons are still needed to detect single molecules since one can only detect the photons with 0.5 - 5% efficiency. 3 The simplest solution is to monitor molecular fluorescence, since the transition can be cycled through 10 5 - 10 6 times 3,11 before photochemical destruction occurs. The second approach is the indirect detection of the molecule of interest. In this scheme, the molecule is cycled through a reaction; i.e., the products from a reaction catalyzed by the molecule of interest are monitored. A catalytic species is
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This note was uploaded on 01/15/2012 for the course CHM 2045 taught by Professor Mitchell during the Fall '07 term at University of Florida.

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8 - Anal. Chem. 1997, 69, 4242-4248 Monitoring the...

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