Templeton D M
Dept. of Clinical Biochemistry, University of Toronto, Canada.
Connect Tissue Res. 1988;17(1):23-32. doi: 10.3109/03008208808992791.
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.
一种基于硫酸化糖胺聚糖与染料1,9 - 二甲基亚甲基蓝（DMMB）反应的快速分光光度法，正得到广泛认可。该检测方法的局限性尚未得到探讨，我们研究了在多种化合物存在下染料的吸收光谱，以了解显色机制。在硫酸化糖胺聚糖存在下，该染料呈现典型的异染性，但似乎是通过比其他噻嗪染料（如天青A）简单得多的机制实现的。有证据支持这样的结论：染料二聚体与聚阴离子反应，通过对发色团的离子扰动产生单一的异染物种。正如所预测的，破坏二聚化的因素，如吡啶和吡啶核苷酸，会妨碍在它们存在时使用该检测方法，并且高盐浓度会在没有聚阴离子的情况下通过诱导异染性而给出假阳性读数。当了解这些因素后，该检测方法可以方便地使用。特别是，它允许直接测量几毫克碱溶性基底膜中的糖胺聚糖含量。
