Resolution of heterogeneous fluorescence from proteins and aromatic amino acids by phase-sensitive detection of fluorescence.

The use of phase-sensitive fluorescence spectroscopy is described for the resolution of heterogeneous fluorescence from a protein and from fluorophore mixtures which model heterogeneous protein fluorescence. This new method permits direct recording of the individual emission spectra of each fluorophore in a two-component mixture or the determination of the lifetime of each species in the mixture. A phase fluorometer is used, modified only by addition of a phase-sensitive detector. To model multi-tryptophan proteins, we examined a mixture of indole and 2,3-dimethylindole. The lifetimes and emission spectra of these fluorophores differ. Choice of the detector phase angle to be out of phase with one component results in a phase-sensitive emission spectrum which precisely superimposes with the steady state emission spectrum of the second unsuppressed component. In addition, heterogeneity of the fluorescence emission is readily revealed by the phase-sensitive emission spectra. For a single compound, or more properly, a single decay rate, the emission maximum is independent of detector phase angle. In contrast a marked dependence on detector phase was observed for the mixture. We also examined a mixture of tyrosine and tryptophan derivatives. In this instance, we used phase-sensitive emission spectra to determine the lifetime of each fluorophore in the mixture. These lifetimes were determined from the detector phase angles necessary to match the phase-sensitive emission spectra with the steady state spectra of either tyrosine or tryptophan. In a similar manner, we recorded directly the emission spectra of the tyrosine or tryptophan residues from denatured human serum albumin and thereby determined the lifetimes of these spectral components. Phase-sensitive fluorescence spectra depend upon both the lifetimes and spectral distributions of each species in a heterogeneous mixture, and thereby provide a practical and powerful approach for the analysis of fluorescence emission from heterogeneous samples. Fluorescence spectroscopic methods are widely used in investigations of proteins, membranes, and other more complex biological systems. Frequently, interpretation of fluorescence data is hindered by the presence of multiple emissions. Several compounds may be fluorescent or a single compound may be present in more than one environment. Examples of heterogeneous emissions are numerous and include fluorescence * This work was supported by Grant PCM 78-16706 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.