Equilibrium and non-equilibrium complexes between bovine serum albumin and dextran sulfate-III. Methylene blue binding by equilibrium complexes

Methylene blue (MB) metachromatic dye binding by bovine serum albumin (BSA) dextran sulfate (DS) complexes at pH 5.6 and ionic strength 0.001 and 0.01 has been studied spectrophotometrically. MB titration with BSA-DS complexes showed that the metachromatic effect (i.e. the maximal for this complex, reduction of the molar extinction coefficient) decreased with increasing protein content in the BSA-DS complex; at the same time the endpoint of MB titration remained practically invariant. This was the basis for a supposition that in BSA-DS complexes there were two types of MB binding sites, namely, A-sites causing metachromasy and B-sites free of such property. A-sites are sulfate groups in protein-free sequences of 5-7 groups and longer. B-sites are possibly protein-free sulfate groups in shorter sequences and BSA anion groups as well. On A-sites MB binding is cooperative. The intrinsic equilibrium constant of MB binding decreases with BSA content growth in the complex. Ionic bonds stabilizing BSA-DS complexes are distributed evenly, i.e. their number per protein molecule is independent of the BSA/DS ratio in the complex.