Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, 570 020, India.
Glycoconj J. 2018 Feb;35(1):129-137. doi: 10.1007/s10719-017-9809-0. Epub 2017 Dec 5.
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.
常规分离、估计和表征糖胺聚糖（GAGs）极具挑战性。此外，分析技术密集，而且通常可用于各种结构、功能和生物学研究的 GAG 数量有限。在这种情况下，可用于估计和阐明二糖组成和种类组成的样品仍然是一个挑战。在本研究中，我们确定了通过变色法估计的分离硫酸化 GAG（sGAG）可以回收用于进一步分析的可行性。在用 DMMB 染料结合测定法估计 sGAG 后形成的 sGAG-DMMB 复合物被去复合物化并回收 sGAG。回收的 sGAG 用醋酸纤维素膜电泳进行分析，并在荧光标记后通过 HPLC 进行二糖组成分析。通过该方案，在所有不同纯度水平的样品中，均观察到通过变色法回收 sGAG 的良好效果。进一步使用醋酸纤维素膜电泳分析表明，sGAG 种之间具有良好的分离效果，即软骨素/硫酸皮肤素和硫酸乙酰肝素，与非硫酸化 GAG 透明质酸的干扰相对较小。通过 HPLC 对回收的 sGAG 特别是肝素硫酸的分析显示出与常规方案获得的 GAG 相似的特征二糖组成。因此，在本文中，我们表明可以在常规估计后以更纯净的形式回收 sGAG，并可用于进一步分析，从而节省宝贵的样品。
