Figure 6. MS and NMR analysis of GAUT13 de novo synthesized products.

(A) MALDI-TOF MS of products synthesized by GAUT13 (1.1 μM) incubated in reaction buffer with UDP-GalA (1 mM) for 3 h and subsequently separated by size exclusion chromatography (SEC) (LH-20 resin). Data shown are for a SEC fraction of the HG enriched for HG of DP 2-9. The observed masses are consistent with HG chains containing one UDP (386 Da, see table inset). (B) Products from (A) (upper panel) analyzed by ¹H NMR and standards of UDP-GalA (second panel), HG DP3 (third panel), and HG mix (DP 7-23) (fourth panel). Peak assignments are: Ribose H1 (~6.0 ppm), Uridine H5 (~5.95 ppm), conjugated anomeric GalA H1 (~5.65 ppm), free reducing α GalA (~5.3-5.35 ppm), and internal GalA (~5-.05-5.10 ppm). The HG trimer contained trace levels of contaminants, indicated by asterisks. The GAUT13 de novo reaction products contained no free reducing α GalA species, while the trimer and HG mix had free reducing α GalA species. (C) Zoomed in region of NMR spectra in (B) of the GAUT13 de novo reaction products and UDP-GalA standard to show the 5.65-5.75 ppm region containing the conjugated anomeric GalA H1. The shoulder present in the GAUT13 de novo reaction products at ~5.7 ppm is indicative of multiple GalA species that are conjugated to UDP, which is absent in the major peak generated from UDP-GalA alone.