Photophysical studies on the interaction of amides with Bovine Serum Albumin (BSA) in aqueous solution: Fluorescence quenching and protein unfolding

Abstract

Addition. of amides containing a HCO(NH2) or CH3CO(NH2) framework to BSA results in a fluorescence quenching. On the contrary, fluorescence enhancement with a shift in the emission maximum towards the blue region is observed on the addition of dimethylformamide (DMF) (HCON(CH3)2). Fluorescence quenching accompanied initially with a shift towards the blue region and a subsequent red shift in the emission maximum of BSA is observed on the addition of formamide (HCO(NH2)), whereas a shift in the emission maximum only towards the red region results on the addition of acetamide (CH3CONH2). Steady state emission spectral studies reveal that amides that possess a free NH2 and N(CH3)2 moiety result in fluorescence quenching and enhancement of BSA respectively. The 3D contour spectral studies of BSA with formamide exhibit a shift in the emission towards the red region accompanied with fluorescence quenching, which indicates that the tryptophan residues of the BSA are exposed to a more polar environment. Circular Dichroism (CD) studies of BSA with amides resulted in a gradual decrease in the α-helical content of BSA at 208nm, which confirms that there is a conformational change in the native structure of BSA. Time-resolved fluorescence studies illustrate that the extent of buried trytophan moieties exposed to the aqueous phase on the addition of amides follows the order DMF<acetamide<formamide and this is attributed to the decrease in the number of hydrogen-bonding sites in alkyl substituted amides. Amides act as a hydrogen-bonding donor and acceptor resulting in a hydrogen-bonding interaction with amino and carboxy moieties (amino acids) present in BSA. The fact that the –NH2 hydrogen and the carbonyl oxygen of amide form a concerted hydrogen-bonding network with the carbonyl oxygen and the amino moieties of amino acids respectively is established from fluorescence methods.