EXTENSIVE LIGAND-PROTEIN COMPLEXATION DETERMINED BY FITTING OF CD DATA - BINDING OF LEVAFIX DYE TO SERUM ALBUMINS

A quantitative approach to the extensive complexation of the reactive Levafix dye to bovine and human serum albumin (BSA and HSA) is describ ed, consisting on processing dichroic (CD) data sets registered over a large wavelength (300-700 nm) and concentration ranges of both protei ns and dye, using an appropriate fitting procedure. This allowed the d etermination of two types of independent binding sites in BSA and of t hree types in HSA. A high (six) number of binding sites pertains to ea ch type of binding site in BSA, while three binding sites are present for each type in HSA. Both the association constant and the CD spectru m of a dye molecule in any binding site of the protein(s) have been ob tained. From these spectral information on the chiral conformation of the ligand bound to the protein has been gained. A relatively high Kuh n's dissimmetry factor is associated to the binding-sites CD, revealin g a rigid, chiral arrangement of the molecule in the complexes with HS A, while the same cannot be assessed with BSA. The investigation has b een then extended to affinity of the dye with HSA precomplexed with ol eic acid. A good correspondence is found in the association constants of the dye with the Cohn fraction V of KSA, and with a purer HSA compl exed with 0.5 mol of oleic acid per mol of protein. Independent suppor t to the results is given by ultrafiltration experiments.