Coumarin 6, Hypericin, Resorufins, and Flavins: Suitable Chromophores for Fluorescence Correlation Spectroscopy of Biological Molecules

Abstract

In this work we show that the dyes coumarin 6, hypericin, 7-O-ethylresorufin and resorufin are suitable for fluorescence correlation spectroscopy (FCS) and demonstrate the use of these dyes in physiologically relevant protein studies. Since coumarins are metabolised by cytochromes P450, the binding of coumarin 6 to cytochrome P450 3A4 was investigated by FCS. Coumarin 6 appears to be a very bright non-covalent cytochrome P450 label. When titrating cytochrome P450 3A4 with coumarin 6, the diffusion time of the coumarin 6/ cytochrome P450 3A4 complex increases roughly two-fold at protein concentrations higher than 1 μmol l-1, indicating the formation of cytochrome aggregates. FCS of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) shows that both endogenous dyes undergo photobleaching. Moreover, FAD appears to be present to great extent, as a non-fluorescent intramolecular complex. Analysis of the FCS data of the flavoprotein NADPH-cytochrome P450 oxidoreductase (molecular weight 76 500) yielded two components. While the slow component corresponds to a globular protein with the molecular weight about 75 000, the fast component appears to be due to free diffusing FMN and FAD molecules. The amount of free FMN and FAD increases with increasing laser power. At high laser power a complete photodissociation of FMN and FAD occurs.