The basis and applicability of the dimethylmethylene blue binding assay for sulfated glycosaminoglycans

A rapid spectrophotometric assay for sulfated glycosaminoglycans, based on reaction of these compounds with the dye 1,9-dimethylmethylene blue (DMMB), is gaining widespread acceptance. The limitations of the assay have not been explored, and we have investigated the absorption spectra of the dye in the presence of a variety of compounds in order to understand the mechanism of colour formation. The dye exhibits classical metachromasia in the presence of sulfated glycosaminoglycans, but appears to do so by a much simpler mechanism than other thiazine dyes such as Azure A. Evidence is offered to support the conclusion that dye dimer reacts with the polyanion to produce a single metachromatic species by ionic perturbation of the chromophore. As predicted, factors which disrupt dimerization, such as pyridine and pyridine nucleotides, preclude use of the assay in their presence, and high salt concentrations can give false positive readings by inducing metachromasia in the absence of polyanion. When these factors are understood, the assay can be used with convenience. In particular, it allows direct measurement of the glycosaminoglycan content of a few mg of alkaline-solubilized basement membrane.