Abstract
Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate and hyaluronic acid. The GAGs are heterogeneous molecules with great variability in molecular mass and both sulfation density and pattern. Spectrophotometric assays to measure the GAG content in biological fluids and tissue/cell extracts are valuable tools. The dye 1,9-dimethylmethylene is a thiazine chromotrope agent that presents a change in the absorption spectrum due to the induction of metachromasia when bound to sulfated GAGs enabling rapid detection of GAGs in solution (Whitley et al., 1989; Chandrasekhar et al., 1987; Farndale et al., 1982). Moreover, there is a window in which a linear curve may be drawn (approximately between 0.5-5 μg of GAGs) enabling the quantification of GAGs in solution.
Keywords: Glycosaminoglycan quantification, Proteoglycans, Sulfated Glycosaminoglycan
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
This protocol was adapted or modified from Farndale et al. (1982) and Whitley et al. (1989).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
I have solved the issue for people who cannot get this assay to work. Simply add 5mL 100% ethanol per litre to the mix OR, within 15 minutes of the assay, add 1 part 2M TRIS per 10 parts DMMB reagent. This is what the TRIS was for.
There are 2 problems with your desire to use this protocol with synovial fluid. First, note 4 indicates it does not with with oligosaccharides shorter than 4 sugars. Hyaluronidase cleaves the polysaccharide into disaccharides. Second, note 5 indicates it doesn't work with non-sulfinated GAGs, such as hyaluronic acid. Looking at the composition of synovial fluid, it seems to only contain hyaluronic acid.Lance
First of all you mentioned your standard curve has also not changed color. That would mean that the DMMB solution would not have been prepared correctly.Second, please read through the notes. Using samples with high salt and albumin content may affect the assay; I am not sure what mouse model you are using to collect your urine samples. Sometimes diluting samples 1:10 can help, especially if you believe the concentration of sulfated GAGs in your samples will fall above the standard curve. You could also add a gel filtration step before the DMMB assay.Also, in the notes we mention that DMMB requires the length of glycosaminoglycan chain be over a tetrasaccharide. GAGs present in the urine are significantly smaller than those present in tissues, so please make sure your samples are appropriate for this assay.
Assuming you media contains L-Cysteine, add EDTA to 0.1 mM and Papain, 0.5 U/mg (Sigma, Cat. No. 76216 - 25 U/ml). Digest samples in papain solution for 15–18 hours at 65°C. Digested samples can be stored at −20°C.
I want to thank you very much for your quick answer!So do you think that the problem is the L-Cysteine and not the FBS? In light of your scientific experience could you briefly explain me why L-Cysteine could be a problem for the GAGs quantification with the DMMB solution?Thank you very much again for your help! It's very much appreciated!Kind regards,MB
Dear Mikhail,I am not sure if I understood the extent of the problem with the standard curve. Were 3, 4 and 5 ng shown in your photo done with the reagent prepared based on our protocol? They seem to have worked. They are very close points on the curve so maybe you could open the curve a little. Use 1.25 microg/mL, 5 microg/mL and 10 microg/mL while testing.Now about your samples, what are they and what were they prepared in? Some detergents or high salts like NaCl and/or Urea can affect the DMB reaction. Could you please provide more information on how these samples were purified?Thank you.
