期刊
JOURNAL OF PHYSICAL CHEMISTRY B
卷 117, 期 5, 页码 1252-1260出版社
AMER CHEMICAL SOC
DOI: 10.1021/jp310663v
关键词
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资金
- Fonds voor Wetenschappelijk Onderzoek FWO [G.0402.09, G.0413.10, G.0697.11]
- K.U. Leuven Research Fund [GOA 2011/03]
- Flemish government [METH/08/04]
- Federal Science Policy of Belgium [IAP-VI/27]
- Hercules Foundation
- Human Frontier Science Program (HFSP)
- Netherlands Organisation for Scientific Research (NWO)
- JSPS
- HFSP
- MEXT
Single molecule fluorescence spectroscopy offers great potential for studying enzyme kinetics. A number of fluorescence reporter systems allow for monitoring the sequence of individual reaction events with a confocal microscope. When using a time-correlated single photon counting (TCSPC) detection scheme, additional information about the fluorescence lifetimes of the fluorophores can be obtained. We have applied a TCSPC detection scheme for studying the kinetics of alpha-chymotrypsin hydrolyzing a double-substituted rhodamine 110-based fluorogenic substrate in a two-step reaction. On the basis of the lifetime information, it was possible to discriminate the intermediate and the final product. At the high substrate concentration used, only the formation of the intermediate was observed. No rebinding of the intermediate followed by rhodamine 110 formation occurred at these high concentrations. We have further found no alterations in the fluorescence lifetime of this intermediate that would indicate changes in the local environment of the fluorophore originating from strong interactions with the enzyme. Our results clearly show the power of using lifetime-resolved measurements for investigating enzymatic reactions at the single molecule level.
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