Identification of the antitumoral drug emodin binding sites in bovine serum albumin by spectroscopic methods.

Using SERS, fluorescence, circular dichroism and stopped-flow, we have unequivocally characterized the binding sites of emodin in bovine serum albumin. Emodin interacts with protein through two different binding sites corresponding to Sudlow's sites 1 and 2. Site 2, where the binding drug presents, in the cavity, a form between neutral and mono-anionic species slightly displaced to the neutral one, is the primary interaction site, with higher association binding constant, and hence, higher affinity than the other binding site. This interaction changes considerably the alpha-helical content of the protein and it occurs mainly within the interval [emodin]/[protein] < or = 2.0. The process involves a fast reaction and the observed rate constant increases when increasing the [emodin]/[protein] ratio. The secondary emodin interaction site corresponds to the Sudlow's site 1, where the drug shows a similar form to that deduced for site 2, but in this case, it is more displaced to mono-anionic species. This interaction does not change the alpha-helical content of bovine serum albumin, and it occurs mainly for [emodin]/[protein] > 2.0 ratios, the process implies a slower reaction than the union process to the site 2, with an observed rate constant that is invariable within the studied interval.