Formycin 5'-triphosphate, a fluorescent analog of ATP, as a substrate for adenylate cyclase.

FluorophoresFluorescence probes represent the most important area of fluorescence spectroscopy. One can spend a great deal of time describing the instrumentation for fluorescence spectroscopy, including light sources, monochromators, lasers, and detectors. However, in the final analysis, the wavelength and time resolution required of the instruments are determined by the spectral properties of the fluorophores. Furthermore, the information available from the experiments is determined by the properties of the probes. Only probes with nonzero anisotropies can be used to measure rotational diffusion, and the lifetime of the fluorophore must be comparable to the correlation time of interest. Only probes which are sensitive to pH can be used to measure pH. And only probes with reasonably long excitation and emission wavelengths can be used with tissues which display autofluorescence at short excitation wavelengths.Thousands of fluorescent probes are known, and it is not practical to describe them all. This chapter contains an overview of the various types of fluorophores, their spectral properties, and their applications. Fluorophores can be broadly divided into two main classes, intrinsic and extrinsic. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of the sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine, and numerous other substances. 3.1.A. Fluorescent Enzyme CofactorsEnzyme cofactors frequently are fluorescent (Figure 3.1). NADH is highly fluorescent, with absorption and emission maxima at 340 and 460 nm, respectively (Figure 3.3). The oxidized form of NADH, NAD+, is nonfluorescent. The lifetime of NADH in aqueous buffer is near 0.4 ns. The 63 J. R. Lakowicz, Principles of Fluorescence Spectroscopy

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Formycin 5'-triphosphate, a fluorescent analog of ATP, as a substrate for adenylate cyclase.

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