SPECTROPHOTOMETRIC STUDIES OF BINDING OF TRYPAN BLUE TO BOVINE SERUM ALBUMIN

An analysis is presented showing that qualitative spectral changes incident to binding can be interpreted in terms of sets of binding sites. A set is defined as a number of occupied sites which are individually indistinguishable spectroscopically, and the minimum number of sets is equal to the number of qualitative spectral changes. Occupied sites will belong to the same set if they have identical spectral properties, identical association constants or if cooperative binding occurs between them. Interactions may alter sites so that a set may cease to exist as additional sites are filled and a new set is formed. The spectra of solutions of the azo dye trypan blue and bovine serum albumin have been measured at pH 7.4 and 3.3 over a wide range of dye [D] to protein [P] ratios. At pH 7.4 the data indicate that there are 2 sets and a total of 3 binding sites. The model most consistent with the results is one in which the stronger set comprises 2 sites, and the filling of the 3rd site involves interactions that result in the disappearance of the stronger set with the formation of the weaker set, which then consists of all 3 sites. At pH 3.3 and high dye to protein ratios and insoluble complex is formed having approximately the composition PD31. At relatively lower dye to protein ratios soluble complexes appear, and the soluble complex in equilibrium with the insoluble one is PD9. There are 3 sets of sites in the soluble complexes, and the 2 strongest sets together consist of at least 5 sites.