Glycosaminoglycans Detection

Glycosaminoglycans (GAGs) are negatively charged polysaccharides located in most connective tissues and extracellular matrix (ECM), as well as on the surfaces of many cell types. Consisting of repeating core disaccharide units, GAGs are categorized into four types: heparin/heparan sulfate, chondroitin/dermatan sulfate, keratin sulfate and non-sulfated hyaluronan. Sulfated GAGs in the ECM exist as proteoglycan which typically consists of multiple glycosaminoglycan chains attached to a core protein. As a highly organized ECM, articular cartilage is composed of type II collagen, hyaluronan, link protein, and chondroitin sulfate-rich proteoglycans, which provide the osmotic resistance necessary for cartilage to resist compressive loads. Chondrex, Inc. provides a sulfated GAGs assay kit using the cationic dye, 1,9 dimethylmethylene blue (DMB) which binds to highly charged sulfated GAGs, not including hyaluronan. This kit utilizes an improved DMB solution minimizing interference with negatively charged contaminants, such as DNA and RNA, and chondroitin sulfate as an appropriate standard for the analysis of ECM in cartilage.

Glycosaminoglycans Assay Kit

Reagents for Sample Preparation

Table 1 provides a useful matrix to determine the ideal solubilization protocol for the analyte of interest.  In most cases, two different protocols will need to be used. For example, protocol A extracts GAGs, but fails to completely extract collagen and DNA, resulting in underestimated values (1,2). Protocol B solubilizes GAGs (3) and DNA (4), but degrades collagen. Finally, protocol C (Chondrex’s protocol) can be used to measure GAGs and collagen (5), but not DNA.

References

1.    C. D. Hoemann, J. Sun, V. Chrzanowski, M. D. Buschmann, A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage. Anal Biochem 300, 1-10 (2002).

2.    S. W. Sajdera, V. C. Hascall, Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J Biol Chem 244, 77-87 (1969).

3.    R. W. Farndale, D. J. Buttle, A. J. Barrett, Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 883, 173-177 (1986).

4.    Y. J. Kim, R. L. Sah, J. Y. Doong, A. J. Grodzinsky, Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 174, 168-176 (1988).

5.    K. von der Mark, M. van Menxel, H. Wiedemann, Isolation and characterization of new collagens from chick cartilage. Eur J Biochem 124, 57-62 (1982).