Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.