Microdetermination of Proteoglycans and Glycosaminoglycans in the Presence of Guanidine Hydrochloride (1)
Proteoglycans are complex macromolecules consisting of a central core protein to which one or more glycosaminoglycans (GAG) are covalently attached.
The DMB reagent was prepared as the following. 16 mg 1,9 dimethylmethylene blue was dissolved in 5 ml ethanol followed by the addition of 2.0 ml formic acid and 2.0 g sodium formate and made up to 1 liter with distilled water. The final concentration of the dye was 12- 14 ug/ml.
The greater the concentration of GuHCl, the lower the sensitivity. For example, in the absence of GuHCl, the absorbance of proteoglycan-DMB complex at a starting amount of 6 ug of proteoglycan is 0.52. However, the absorbance was reduced to 0.32 at 0.16 M GuHCl or to 0.13 (at 0.32 M GuHCl). All interactions were completely abolished at 0.8 M GuHCl. In the absence of GuHCl, and in the presence of up to 4 ug/ml of proteoglycan or DNA, the interaction is indistinguishable. However, at higher concentrations, the DNA-DMB complex precipitated out of the solution resulting in decreased absorbance.
DNA (up to 4 ug/well) neither increased nor decreased the absorbance of proteoglycan-dye interaction. However, at a higher concentration of DNA (10 ug/well) the absorbance was decreased.
All the GAGs can be quantitatively determined, in either the presence or absence of 0.24 M GuHCI. Hyalur- onic acid, an unsulfated GAG, and other matrix glycoproteins such as fibronectin and type II collagen did not interact with the dye in the presence of GuHCl, although minimal interaction was observed for hyaluronic acid in the absence of GuHCl.
The cartilage proteoglycan bound the best. The proteoglycans from bone (chondroitin sulfate), cornea (dermatan + keratan sulfate), and sclera (keratan sulfate) also interacted with the dye but to a much lesser extent, due to the relatively reduced GAG content.
This assay is dependent only on the sulfated GAGS. The cartilage proteoglycan has much higher carbohydrate content (70-80%) than the proteoglycans from scleral (50-60%), cornea (50-55%), or bone (55-70%).
Several dyes including toluidine blue, methylene blue, saffronin-O, Stainsall, and Alcian blue, which are used for histochemical techniques, were tested but dimethylmethylene blue was the most reliable and reproducible. Inclusion of GuHCl or urea (up to 0.40 M; data not shown) in the assay, helps to stabilize the proteoglycan-dye interaction and therefore the complex is stable for a longer time. Finally, the interference of other anionic material such as nucleic acids is either reduced or abolished, thus making the assay more specific.