Supplementary Figure 5: ESI-MS analysis and kinetics assay control to test for donor hydrolysis.
From: Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation
ESI-MS analysis of (a) axial CMP-3FNeu5Ac alone and (b) after 4 day incubation with ST8SiaIII Δ80. Data were recorded in the negative mode with dilutions ranging one order of magnitude. (c) CMP release (coupled to NADH oxidation measured by absorbance at 340 nm) using the assay conditions as described in methods, both before (in unfilled squares) and after (in filled circles) addition of ST8SiaIII to 1 μM. CMP-SA concentration was fixed at 1 mM and SiaLacNAc-6S concentration was varied. At 0 SiaLacNAc-6S, when the enzyme is added, there is no difference in the rate compared to the background control measurements before the enzyme is added. The background rate likely arises from spontaneous oxidation of NADH.
