Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×10⁴ at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583