Binding of bromosulphthalein to serum albumins.

The binding of bromosulphthalein to human and bovine Serum albumin was studied by infrared spectroscopy, laser-Raman spectroscopy, visible spectroscopy and pH measurements in order to obtain information on the binding forces involved. No conformational change of the proteins was observed during the tight binding of the first three bromosulphthalein molecules as indicated by the kinetics of the H-ZH exchange and infrared spectroscopy in 2Hz0. Subsequent occupation of the low affinity binding sites causes a partial unfolding of the proteins. Binding of the dye at the high affinity sites is accompanied by a change in intensity and a shift of the lactone carbonyl band in infrared and laser-Raman spectra as well as a decrease of the visible absorption at 580 nm suggesting a hydrophobic environment. Binding at these sites is caused by Van der Waal or hydrophobic forces since the charge of the proteins remains unchanged during this process. It may be concluded that the main binding forces at the 14 low affinity binding sites consist of electrostatic interactions as indicated by pH shift studies and model studies for the bathochromic shift of the quinoic dye.