[Spectroscopic and thermodynamic investigations on the binding of azure B to chondroitin sulfate and the structure of the metachromatic dye complex]

Abstract

The binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO-4 and COO(-)-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric titration of CHS with azure B, while the SO-4 and COO- content of CHS was determined by conductometric titration of CHS-acid with NaOH. The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0 less than r less than 0.8 (r = the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn = 2.5 X 10(3) M-1) and aggregation (Kq = 1.2 X 10(5) M-1), as well as the cooperativity parameter (q = 50), T = 295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (delta Hoq = -20.0 kJ mol-1) and entropy (delta Soq = 29.7 JK-1 mol-1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions. Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic - the more birefringent, the better the mechanical orientation.(ABSTRACT TRUNCATED AT 400 WORDS)