Fatty acid binding to bovine serum albumin prevents formation of intermediate during denaturation.

Urea-induced denaturation of defatted BSA and BSA containing six mol palmitic acid/mol protein (fatted BSA) has been studied by the techniques of UV difference spectroscopy and fluorescence spectroscopy. The notable differences between the two transitions are a shift in the transition of fatted BSA to a higher urea concentration and a three-state denaturation transition in defatted BSA compared to an apparent two-state denaturation transition in fatted BSA. The stable denaturation intermediate in defatted BSA occurs at 4.5-5.0 M urea, a urea concentration at which denaturation in fatted BSA has not yet started. These results are further supported by the difference spectral results obtained at 4.5 M urea in the two albumin preparations. The occurrence of denaturation intermediate only in defatted BSA and the presence of the two strong fatty-acid-binding sites in domain III lead us to conclude that this domain is relatively unstable in the absence of fatty acids and is responsible for the formation of intermediate.