Consequences of inhomogeneous broadening on fluorescence line narrowing spectra

Kaposi, András Dezső and Wright, W. W. and Vanderkooi, J. M. (2003) Consequences of inhomogeneous broadening on fluorescence line narrowing spectra. JOURNAL OF FLUORESCENCE, 13 (1). pp. 59-67. ISSN 1053-0509

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Abstract

Fluorescence line narrowing is a sensitive tool to monitor subtle changes in protein conformation. We restudied the consequences of inhomogeneous broadening on the observed spectra. Vibronic absorption bands of systems with weak electron-phonon coupling can be described by a lorentzian function with width of the order of a few centimeters. For an ensemble of molecules with closely spaced electronic transitions, a single laser frequency will excite many molecules through the broad base ("tail") of the lorentzian function. This, along with excitation of low-frequency modes, contributes to the unresolved background observed in line-narrowed spectra. Examples are shown for Zn cytochrome c, a fluorescent derivative of Fe cytochrome c. Spectra are compared for the protein in two solvents: glycerol/water or trehalose. Two types of cytochrome, from horse and yeast, are also compared.

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