Characterization of Glycosaminoglycans by 15N-NMR Spectroscopy and in vivo Isotopic Labeling

摘要

Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of ¹H and ¹³C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on ¹H-¹⁵N two dimensional NMR experiments is described. The ¹⁵N- and ¹H-chemical shifts of amide signals from ¹⁵N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6- or 4,6-), and easily distinguishable from those of DS. The amide signals from residual ¹⁵N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of ¹⁵N despite its low percentage. However enrichment of the ¹⁵N-content in GAGs using metabolic incorporation from ¹⁵N-glutamine added to cell culture media is also demonstrated, and used to distinguish metabolic states in different cell types.