Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue (1)
Dimethylmethylene blue assay was subject to positive interference by poly-anions other than sulphated glycosaminoglycans, including hyaluronic acid, DNA and RNA.
The colour reagent was prepared by dissolving 16 mg dimethylmethylene blue in 1 water containing 3.04 g glycine, 2.37 g NaCl and 95 ml 0.1 M HCl, to give a solution at pH 3.0. 100 ul of each sample, containing up to 5ug glycosaminoglycan was placed in a polystyrene tube and the 2.5 ml dimethylmethylene blue colour reagent was added. Mixing was completed by pouring the solution into a disposable spectrophotometer cuvette, nd A525 was read immediately, or after a short, fixed time.
Standard curves for chondroitin sulphate, keratan sulphate, hyaluronic acid and DNA are shown. The slopes for the sulphated glycosaminoglycans were much the same as under the old conditions. In contrast to our previous results, the colour yields of hyaluronic acid and DNA were negligible under the new conditions. This is attributed to suppression of their relatively weak interaction with dimethylmethylene blue by the lower pH and higher salt concentration of the new reagent. The digestion with papain was necessary to eliminate interference by proteins or glycoproteins. The interference could be either positive or negative. The difference spectrum due to the presence of chondroitin sulphate in the reaction mixture showed the major positive peak is at 525 nm and negative spectral change occurred at 590 nm.
The system in which A525 is read is unstable; sulfated glycosaminoglycan- dimethylmethylene blue complexes start to aggregate, and eventually to precipitate, as soon as the glycosaminoglycan and dye are mixed. This is seen as a slow, progressive fall in A525 during the first 10 min or so, eventually followed by gross precipitation. Specific polysaccharide-degrading enzymes are very easily used with the dimethylmethylene blue method. These enzymes degrade glycosaminoglycans efficiently to small oligosaccharides that give no colour reaction with dimethylmethylene blue. The polysaccharidases were selective and at least 89% effective in eliminating the colour reactions of the corresponding glycosaminoglycans.
Reaction with dimethylmethylene blue was monitored by assaying 100 ul samples of the incubation mixture every 10 min, and standardization was with a chondroitin 4-sulphate standard curve for the first 60 min, and a keratan sulphate curve subsequently. The chondroitin 4-sulphate in the mixture was accurately determined and 88% of the keratan sulphate was also accounted for. The use of the polysaccharide lyases in conjunction with the dimethylmethylene blue assay can give valuable estimates of the amounts of the individual glycosaminoglycans, chondroitin sulphate, dermatan sulphate and keratan sulphate.
