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One-pot analysis of sulfated glycosaminoglycans

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Abstract

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

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Fig. 1: Schema for processing of various biological samples.
Fig. 2: sGAG recovery protocol.
Fig. 3: Spectral analysis of sGAG-DMMB complex and recovery of sGAG after decomplexation.
Fig. 4: Recovery of commercially available standard sGAGs after DMMB assay.
Fig. 5: Cellulose Acetate Membrane Electrophoresis of sGAGs from Biological samples.
Fig. 6: HS Disaccharide composition analysis of DG and CG-DX isolated from kidney.

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Abbreviations

CS:

Chondroitin sulfate

DS:

Dermatan sulfate

GAGs:

Glycosaminoglycans

HA:

Hyaluronic acid

HS:

Heparan sulfate

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Acknowledgements

The authors acknowledge the funding of this study by SERB, DST, New Delhi (EMR/2015/000028) and Director for the keen interest. Shrikanth thanks CSIR for the Senior Research Fellowship.

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  1. C. B. Shrikanth and J. Sanjana contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, 570 020, India

    C. B. Shrikanth, J. Sanjana & Nandini D. Chilkunda

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  1. C. B. Shrikanth
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  2. J. Sanjana
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CBS and SJ did experiments, analysed data and wrote manuscript. CDN conceived the work, analysed data and wrote manuscript.

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Correspondence to Nandini D. Chilkunda.

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Shrikanth, C.B., Sanjana, J. & Chilkunda, N.D. One-pot analysis of sulfated glycosaminoglycans. Glycoconj J 35, 129–137 (2018). https://doi.org/10.1007/s10719-017-9809-0

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