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Dimethylmethylene Blue Assay (DMMB)
二甲基亚甲基蓝试验(DMMB)

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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(糖胺聚糖定量), Proteoglycans(蛋白多糖), Sulfated Glycosaminoglycan(硫酸化糖胺聚糖)

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.

简介

糖胺聚糖(GAG)是长的非支链多糖,由由己糖胺(葡糖胺或半乳糖胺)和己糖醛酸(葡糖醛酸或艾杜糖醛酸)组成的重复二糖单元组成。根据二糖单位,GAG可以分为五组:硫酸软骨素,硫酸皮肤素,硫酸乙酰肝素,硫酸角质素和透明质酸。 GAG是具有分子质量和硫酸化密度和模式的大变异性的异质分子。用于测量生物流体和组织/细胞提取物中GAG含量的分光光度测定法是有价值的工具。染料1,9-二甲基亚甲基是噻嗪变体试剂,其由于当与硫酸化GAG结合时诱导变态变化而呈现吸收光谱的变化,使得能够快速检测溶液中的GAG(Whitley等人, ,1989; Chandrasekhar et al。,1987; Farndale et al。,1982)。此外,存在其中可绘制线性曲线(大约在0.5-5μgGAG之间)的窗口,使得能够定量溶液中的GAG。

关键字:糖胺聚糖定量, 蛋白多糖, 硫酸化糖胺聚糖

材料和试剂

  1. 二甲基亚甲蓝(DMMB)(Sigma-Aldrich,目录号:341088)
  2. NaCl
  3. 甘氨酸(Sigma-Aldrich,目录号:410225)
  4. 冰醋酸(Sigma-Aldrich,目录号:S7653)
  5. Tris-Base(Merck KGaA,目录号:648310)
  6. 标准的牛软骨素4-硫酸盐(Sigma-Aldrich,目录号:C9819)
  7. DMMB试剂(参见配方)

设备

  1. 板混合器(VWR International,目录号:89202-332)
  2. 盖粘合剂(R& D Systems,目录号:DY992)
  3. 具有525nm的酶标仪(BioTek Instruments,目录号:11-120-531)
  4. 96孔微孔板分光光度计,具有525nm滤光片组(Thermo Fisher Scientific,目录号:51119200)
  5. 微孔板摇动器(VWR International,目录号:97043-608)

程序

  1. 使用Whattman ® 3MM制备DMMB试剂和滤纸。 该溶液的pH为约3.0。 为了制备1L染料溶液,将16mg DMMB溶解在含有3.04g甘氨酸,1.6g NaCl和95ml 0.1M乙酸的1L水中。
  2. 制备软骨素4硫酸盐(500μg/ml在H 2 O中)的标准溶液。 按下表所述准备标准曲线。
  3. 吸取标准储备溶液,并用H 2 O 2将体积补至20μl,加入96孔微板中。
  4. 吸取20μl的每个样品到微孔板中
  5. 向每个样品中加入200μlDMMB,并将板振荡器的板摇动5秒
  6. 立即用读板仪在525nm处读取吸光度。

    Std(μg/ml)
    500μg/ml std的量(μl)
    vol H 2 O(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

笔记

  1. DMMB测定通常可以对具有高洗涤剂和盐浓度的样品进行; 然而,标准曲线应在同一解决方案中准备
  2. 避免对可能干扰测定的高白蛋白或血清浓度的样品进行测定(Warren,2000)
  3. 一些研究小组报道了DNA在DMMB测定中的干扰,然而,将pH降低至约3并且增加盐浓度使得DNA的干扰可忽略。
  4. DMMB需要糖胺聚糖链的长度超过四糖
  5. DMMB与GAG链的硫酸基反应,因此不会与未硫酸化的GAG(例如透明质酸)一起使用。
  6. 通过使用咔唑反应(灵敏度为1至20μg)可以获得进一步的信息,以测定硫酸肝素,硫酸软骨素和硫酸皮肤素的糖醛酸的羧基和/或蒽酮反应以测定硫酸角质素的己糖基团 Mort和Roughley,2007)。
  7. 当进行色谱法以快速测定含有GAG的级分时,DMMB也可以是非常有效的(Burton-Wurster等人,2003)。
  8. 形成的DMMB-GAG复合物导致立即形成浑浊,然而这种复合物在10分钟内开始沉淀,因此应立即进行吸光度测量。

食谱

  1. DMMB试剂
    溶解16mg DMMB,3.04g甘氨酸,1.6g NaCl和95ml 0.1M乙酸,并使体积达到1L。
    过滤器(0.45μm)
    避光
    如果溶液中存在沉淀,请勿使用

致谢

该方案适应或修改自Farndale等人(1982)和Whitley等人(1989)。

参考文献

  1. Anwari,K.,Webb,CT,Poggio,S.,Perry,AJ,Belousoff,M.,Celik,N.,Ramm,G.,Lovering,A.,Sockett,RE,Smit,J.,Jacobs-Wagner ,C。和Lithgow,T。(2012)。 细菌外膜BAM复合物的新脂蛋白亚基的进化 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)。 TGFβ1和犬软骨中的双链蛋白聚糖,核心蛋白多糖和纤维蛋白的代谢。 Osteoarthritis Cartilage 11(3):167-176。
  3. Chandrasekhar,S.,Esterman,M.A。和Hoffman,H.A。(1987)。 在胍盐酸盐存在下微量测定蛋白多糖和糖胺聚糖。 Anal Biochem 161(1):103-108。
  4. Farndale,R.W.,Sayers,C.A。和Barrett,A.J。(1982)。 用于软骨培养中硫酸糖胺聚糖的直接分光光度法微量测定。连接Tissue Res 9(4):247-248。
  5. Mort,J.S。和Roughley,P.J。(2007)。 测量从软骨外植体释放的糖胺聚糖。 Methods Mol Med 135:201-209。
  6. Warren,S。(2000)。 1,9-二甲基亚甲基蓝的关键分析 。。
  7. Whitley,C.,Ridnour,M.,Draper,K.,Dutton,C。和Neglia,J。(1989)。 粘多糖病的诊断测试。 I.用于定量过量尿糖胺聚糖排泄的直接方法。临床化学 35(3):374-379。
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引用:Coulson-Thomas, V. . and Gesteira, T. F. (2014). Dimethylmethylene Blue Assay (DMMB). Bio-protocol 4(18): e1236. DOI: 10.21769/BioProtoc.1236.
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Matteo Beretta
Fondazione Ca' Granda - Ospedale Maggiore Policlinico
Dear Sir, Dear Madame,

I tried to use your protocol for GAGs quantification in cell colture medium conditioned by intervertebral disc cells for 24h-48h and used the bovine chondroitin 4-sulfate as standard. I am having huge problems even with the standard curve (a eight point-curve in the range 10 ug/ml - 0 ug/ml) since the curve appears linear when prepared with H2O, whereas the machine read almost the same absorbance value at 252nm when the curve is prepared with DMEM+10%FBS or DMEM+0.5%FBS.
Could you give me your opinion about this problem and any possible explanation/solution to that, please?

P.S. I saw that some authors use to digest their samples with papain before the GAGs quantification. Could it help me somehow? And in case yes, do you recommend a particular papain solution and digestion time?

I thank you very much in advance and wish you all a good day.

Kind regards,

MB
6/22/2017 4:53:20 AM Reply
Tarsis Gesteira
Department of Ophthalmology, University of Cincinnati, USA

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.

6/22/2017 4:01:18 PM


Matteo Beretta
Fondazione Ca' Granda - Ospedale Maggiore Policlinico

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

6/23/2017 2:04:34 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
2/23/2016 12:34:28 AM Reply
Vivien Coulson-Thomas
College of Optometry, the Ocular Surface Institute (TOSI), University of Houston, USA

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.



3/2/2016 9:10:38 AM


Mahadevan R
Dept of Biochemistry
Sir, can I use this method to detect the sulfated GAG in a decellularized ECM? For that do I have to extract the sulfated GAG first or can I dissolve the decellularized ECM in

Sir, I have extracted collagen from fish skin. It has a
10/14/2015 2:17:29 AM Reply