Optical studies on the molecular-sterical mechanism of metachromasia.

We have investigated how the stereochemical situation of the dye binding negative side groups on polysaccharides influences metachromatic staining effects. We found that selective binding of toluidine blue (pH = 1.0) in the places of substituted (acidified) vicinal OH groups C2 and C3 resulted in the clearest metachromasia with the strongest anisotropy effects and with a bright clear green polarization color. On the other hand, selective toluidine blue binding in the places of C6 of the carbohydrate rings of the polysaccharide chains resulted in orthochromatic basophilia and lack of birefringence. After transformation of all OH side groups into negatively charged groups, metachromatic staining was found mixed with an orthochromatic hue and a birefringence effect weaker than in the case of staining on the vicinal OH groups only. The need of poststaining stabilization of the labile metachromatic staining reactions is emphasized.