Benzo[a]pyrene--serum : albumin/cysteine interactions: fluorescence and electron spin resonance studies

The complex between bovine serum albumin (BSA) and benzo[a]pyrene (BaP) exhibits three different types of fluorescence. The visible fluorescence at 407, 431 and 458 nm is modified by the formation of both hydroxy-BaP derivatives and BaP products strongly bound to the protein. The ultraviolet fluorescence I is characterized by a triple-peaked structural fluorescence with maxima at 340, 357 and 378 nm. Upon addition of mercaptoethanol, this ultraviolet fluorescence decreases. The ultraviolet fluorescence II appears at 380 nm and corresponds to that of pyrene-like products. When irradiated with ultraviolet (365 nm) light, both the ultraviolet fluorescence I and the visible fluorescence decreases. In the interactionsof BaP with BSA, a new radical has also been found in addition to the known 6-oxo-BaP radical. The lyophilized BSA-BaP complex exhibits two broad ESR bands, one of which increases in lipid-free BSA. In the concentrated aqueous solution of the BSA-BaP complex, the ESR signal is converted to a six-line spectrum. The benzene extract, which removes non-covalently bound BaP products, shows an ESR signal similar to that in aqueous solution except for the absence of two lower g-valued lines. When irradiated with ultraviolet (365 nm) light the signal intensity of the new radical species decreases, while that of 6-oxo-BaP increases. Upon addition of of mercaptoethanol, the signal of the new radical also diminishes and is replaced by a single narrow signal. Ths mixture of BaP and cysteine kept at room temperature for one day in the dark produces both a six-line ESR spectrum and a broad ultraviolet fluorescence at 330 nm, which gradually decay in about one week. When BaP and cysteine are mixed at 65 degrees C for several hours, little ultraviolet fluorescence and ESR signal are detected. The results indicate the formation of a BaP radical as an intermediate in the interaction of BaP with BSA and cysteine in the presence of oxygen and suggest the involvement of SH-groups in this interaction.