Figure 2. Development of a novel microfluidic mobility shift assay to quantify HS2ST enzymatic activity.

(A) Schematic showing PAPS-dependent sulfate incorporation into the fluorescein-labelled hexasaccharide IdoA substrate by HS2ST, with the concomitant generation of PAP. R = fluorescein. (B) NMR analysis of the non-sulfated and sulfated hexasaccharides. The addition of a 2-O-sulfate group to the iduronate (l-IdoA) residue of the fluorescent hexasaccharide results in a significant chemical shift change, most notably to the anomeric proton (H-1) and that of H-2 attached to the sulfated carbon atom of l-IdoA, in agreement with expected values from the literature [47]. ¹H NMR spectrum of non-sulfated substrate (bottom spectrum, black) and sulfated product (upper spectrum, red). Distinct l-IdoA protons (H-3 and H-4 of the spin system) were identified by TOCSY and are shown vertically above their respective H-1 signals (for the non-sulfated substrate, right blue boxed, and for the sulfated product, left blue boxed). The full carbohydrate proton spectra are shown in Supplementary Figure S3. (C and D) Screen shots of EZ reader II raw data files, demonstrating that HS2ST induces a rapid mobility change in the IdoA-containing fluorescent hexasaccharide. Separation of the higher mobility, sulfated (product, P) from the lower mobility (substrate, S) hexasaccharide occurs as a result of enzymatic substrate sulfation (left panels 180 s assay time, right panels 240 s assay time), as demonstrated by omission of HS2ST from the assay (−HS2ST). Assays were initially performed at 20°C using 90 nM of purified HS2ST, 2 μM fluorescein-labelled hexasaccharide substrate and 500 μM PAPS. (E) Stoichiometric sulfate-labelling of IdoA-containing fluorescein-labelled hexasaccharide. Reactions were performed with 0.6 μM HS2ST, 375 μM IdoA-hexasaccharide substrate and 1 mM PAPS and incubated at room temperature for 48 h. The reaction was spiked with an additional 0.5 mM (final concentration) of PAPS after 24 h of incubation. M = non-sulfated marker substrate. A final hexasaccharide concentration of 2 μM was analysed by the fluorescent sulfation mobility assay. (F) Analysis of time-dependent sulfate incorporation into 2 μM IdoA-containing fluorescein-conjugated hexasaccharide. Percentage sulfation was calculated from the ratio of substrate hexasaccharide to product (2-O-sulfo)-hexasaccharide at the indicated time points in the presence or absence of 20 nM HS2ST and 10 μM PAPS. (G) Calculation of K_m [PAPS] value for HS2ST. PAPS concentration was varied in the presence of a fixed concentration of HS2ST (20 nM), and the degree of substrate sulfation calculated from a differential kinetic analysis, n = 2 assayed in duplicate. (H) Duplicate HS2ST assays conducted in the presence of increasing concentrations of activating Mg²⁺ ions. Activity is presented in duplicate relative to buffer controls. Similar results were seen in several independent experiments.