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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.

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Dimethylmethylene Blue Assay (DMMB)

Biochemistry > Carbohydrate > Glycoprotein
Authors: Vivien Jane Coulson- Thomas
Vivien Jane Coulson- ThomasAffiliation: John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK
For correspondence: vcoulsonthomas@gmail.com
Bio-protocol author page: a1653
 and Tarsis Ferreira Gesteira
Tarsis Ferreira GesteiraAffiliation 1: Department of Ophthalmology, University of Cincinnati, Cincinnati, USA
Affiliation 2: Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, USA
Bio-protocol author page: a1654
Vol 4, Iss 18, 9/20/2014, 12807 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1236

[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

  1. Dimethylmethylene blue (DMMB) (Sigma-Aldrich, catalog number: 341088)
  2. NaCl
  3. Glycine (Sigma-Aldrich, catalog number: 410225)
  4. Glacial acetic acid (Sigma-Aldrich, catalog number: S7653)
  5. Tris-Base (Merck KGaA, catalog number: 648310)
  6. Bovine chondroitin 4-sulfate as standard (Sigma-Aldrich, catalog number: C9819)
  7. DMMB reagent (see Recipes)

Equipment

  1. Plate mixer (VWR International, catalog number: 89202-332)
  2. Cover adhesive (R&D Systems, catalog number: DY992)
  3. Microplate reader with 525 nm (BioTek Instruments, catalog number: 11-120-531)
  4. 96 well microplate spectrophotometer with 525 nm filter set (Thermo Fisher Scientific, catalog number: 51119200)
  5. Microplate shaker (VWR International, catalog number: 97043-608)

Procedure

  1. Prepare DMMB reagent and paper filter using Whattman® 3MM. The pH of this solution is around 3.0. To prepare 1 L dye solution, dissolve 16 mg DMMB in 1 L water containing 3.04 g glycine, 1.6 g NaCl and 95 ml of 0.1 M Acetic Acid.
  2. Prepare standard solution of chondroitin 4 sulfate (500 µg/ml in H2O). Prepare standard curve as stated in the table bellow.
  3. Pipet the standard stock solution and complete the volume to 20 µl with H2O into the 96 well microplate.
  4. Pipet 20 µl of each sample into the microplate.
  5. Add 200 µl of DMMB to each sample and shake the plate of a plate shaker for 5 sec.
  6. Read the absorbance using a plate reader at 525 nm immediately.

    Std (µg/ml)
    Vol (µl) of 500 µg/ml std
    vol H2O (µl)
    vol DMMB (µl)
    0
    0
    20
    200
    1.25
    2.5
    17.5
    200
    2.5
    5
    15
    200
    5
    10
    10
    200
    7.5
    15
    5
    200
    10
    20
    0
    200

Notes

  1. DMMB assay can normally be performed on samples with high detergent and salt concentrations; however the standard curve should be prepared in the same solution.
  2. Avoid to performing the assay on samples in high albumin or serum concentrations which may interfere with the assay (Warren, 2000).
  3. Some groups have reported the interference of DNA in the DMMB assay, however, decreasing the pH to approximately 3 and increasing salt concentrations makes the interference of DNA negligible.
  4. DMMB requires the length of glycosaminoglycan chain be over a tetrasaccharide.
  5. DMMB reacts with the sulfate group of the GAG chain and therefore will not work with unsulfated GAGs such as hyaluronic acid.
  6. Further information can be acquired by utlilizing the carbazole reaction (sensitivity from 1 to 20 μg) to assay the carboxyl groups of the uronic acid for heparin sulfate, chondroitin sulfate and dermatan sulfate and/or anthrone reaction to assay the hexose group for keratan sulfate (Mort and Roughley, 2007).
  7. The DMMB can also be very efficient when performing chromatography to rapidly assay for fractions containing GAGs (Burton-Wurster et al., 2003).
  8. The DMMB-GAG complex that is formed results in the immediate formation of turbidity, however this complex starts to precipitate within 10 min, therefore the absorbance measurement should be performed immediately.

Recipes

  1. DMMB reagent
    Dissolve 16 mg DMMB, 3.04 g glycine, 1.6 g NaCl and 95 ml of 0.1 M acetic acid and complete the volume to 1 L
    Filter (0.45 μm)
    Protect from light
    Do not use if precipitate is present in the solution

Acknowledgments

This protocol was adapted or modified from Farndale et al. (1982) and Whitley et al. (1989).

References

  1. Anwari, K., Webb, C. T., Poggio, S., Perry, A. J., Belousoff, M., Celik, N., Ramm, G., Lovering, A., Sockett, R. E., Smit, J., Jacobs-Wagner, C. and Lithgow, T. (2012). The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex. Mol Microbiol 84(5): 832-844.
  2. Burton-Wurster, N., Liu, W., Matthews, G. L., Lust, G., Roughley, P. J., Glant, T. T. and Cs-Szabo, G. (2003). TGF beta 1 and biglycan, decorin, and fibromodulin metabolism in canine cartilage. Osteoarthritis Cartilage 11(3): 167-176.
  3. Chandrasekhar, S., Esterman, M. A. and Hoffman, H. A. (1987). Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal Biochem 161(1): 103-108.
  4. Farndale, R. W., Sayers, C. A. and Barrett, A. J. (1982). A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9(4): 247-248.
  5. Mort, J. S. and Roughley, P. J. (2007). Measurement of glycosaminoglycan release from cartilage explants. Methods Mol Med 135: 201-209.
  6. Warren, S. (2000). A critical analysis of the 1, 9-dimethylmethylene blue assay for sulfated glycosaminoglycans in Synovial fluid. University of Guelph.
  7. Whitley, C., Ridnour, M., Draper, K., Dutton, C. and Neglia, J. (1989). Diagnostic test for mucopolysaccharidosis. I. direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem 35(3): 374-379.


How to cite this protocol: Coulson-Thomas, V. . and Gesteira, T. F. (2014). Dimethylmethylene Blue Assay (DMMB). Bio-protocol 4(18): e1236. DOI: 10.21769/BioProtoc.1236; Full Text



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2/23/2016 12:34:28 AM  

Mikhail Olinevich
Biomed

Dear sir/madame,

I've followed the exact protocol to find sulfated glycosaminoglycans in human skin samples which have been degraded using pronase. A kit from "Biocolour, Blyscan" quantifies the sGAGs in our sample in a proper way. Because of its expensiveness, we have decided to make the dimethylmethyleneblue reagens by ourselfs. The problem that we are experiencing is that no precipitate occurs. By adding some leftovers of the dye reagent from Blyscan, the precipitate immediately follows. How could you explain this problem? Why isn't it working? I suppose it has to do something with the reagent...
The second problem is that there is no variation in absorbance between the different standard concentrations. The absorbance reader (tried at 540 and 510 nm) gives approximately the same value for absorbance for different concentration of chondroitin-4S standard (see photo attachment). I suppose, this problem is also linked to the first one.

At last, in material and reagents, you mention that you need Tris base, but you actually don't use it in the protocol..

Thank you in advance for you help.
Mikhail

   
3/2/2016 9:10:38 AM  

Vivien Coulson-Thomas (Author)
John van Geest Centre for Brain Repair,University of Cambridge

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.



Reply

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