FATTY-ACID-BINDING TO BOVINE SERUM-ALBUMIN PREVENTS FORMATION OF INTERMEDIATE DURING DENATURATION

Urea-induced denaturation of defatted BSA and BSA containing six mol p almitic acid/mol protein (fatted BSA) has been studied by the techniqu es of UV difference spectroscopy and fluorescence spectroscopy. The no table differences between the two transitions are a shift in the trans ition of fatted BSA to a higher urea concentration and a three-state d enaturation transition in defatted BSA compared to an apparent two-sta te denaturation transition in fatted BSA. The stable denaturation inte rmediate in defatted BSA occurs at 4.5-5.0 M urea, a urea concentratio n at which denaturation in fatted BSA has not yet started. These resul ts are further supported by the difference spectral results obtained a t 4.5 M urea in the two albumin preparations. The occurrence of denatu ration intermediate only in defatted BSA and the presence of the two s trong fatty-acid-binding sites in domain III lead us to conclude that this domain is relatively unstable in the absence of fatty acids and i s responsible for the formation of intermediate.