How to measure Glycosaminoglycans and Proteoglycans?
Glycosaminoglycans (or mucopolysaccharides) are long un-branched polysaccherides which consist of disaccharide repeats. The repeats usually (with the exception of Keratan sulfate) consist of on amino sugar (N-acetylgalactosamine of N-acetylglucosamine) and a uronic sugar (iduronic acid or glucuronic acid) or galactose.
Glycosaminoglycans are highly polar and function as water attractants, thus they are useful as shock absorbers or lubricants.
Four glycosaminoglycan classes can be distinguished:
- Heparin/heparan sulfate
- Chondroitin sulfate/dermatan sulfate
- Keratan sulfate
- Hyaluronic acid
These structures differ in their core disaccaride structure and their synthesis pathways. While Heparin/heparan sulfate, Chondroitin sulfate/dermatan sulfate and Keratan sulfate are produced by the Golgi apparatus, Hyaluronic acid is synthezised by integral membrane synthases which immediately secrete the elongated disaccharide chain.
Proteoglycans – core proteins attached to glycosaminoglycans
Proteoglycans consist of core proteins which are covalently linked to glycosaminoglycans – either mono- or poly-glycosylated. The glycosaminoglycan is coupled though a tetrasaccharide bridge to a Serin residue of the protein. Proteoglycans represent the major component of the animal extracellular matrix where they form complex networks with other proteoglycans and fibrous matrix proteins such as collagen. These networks are involved in binding water and diverse cations.
Mucopolysaccharidoses are genetic discorders which lead to the inability to degrade proteoglycans. As a consequence an accumulation of proteoglycans occurs which subsequently leads to a number of pathological symptoms (dependent on the type of proteoglycan which cannot be degraded).
One assay to measure almost all glycosaminoglycans and peptidoglycans
The Blyscan Assay (see flowchart Fig. 1) is a quantitative dye-binding method for the analysis of sulfated proteoglycans and glycosaminoglycans. Test material can be assayed directly when present in a soluble form, or following papain extraction from biological materials. The assay can be used to measure the total glycosaminoglycans content and can also be adopted to determine the O- and N-sulfated glycosaminoglycan ratio within test samples.
The dye label used in the assay is 1, 9-dimethylmethylene blue and the dye is employed under conditions that provide a specific label for the sulfated polysaccharide component of proteoglycans or the protein free sulfated glycosaminoglycan chains.
Note: the assay is not suitable for small sulfated disaccharide fragments or for samples containing alginates, as these contain uronic acid.
For analysing which soluble extracts?
- fibrous and hyaline cartilages
- arteries, heart, lung, skin and other material containing extracellular matrix, (connective tissue) and solid tumours (results see Fig. 2)
- extracellular matrix components that may be released by live cells into the culture medium, some of which can be attached to cell culture plasticware
- soluble glycosaminoglycans from synovial fluid, urine and gel chromatography fraction aliquots
Would the Blyscan assay be of use in your research? Share your comments or questions below!