Fig. 3.
A) Purification of C5-epimerase on a heparin-Toyopearl affinity chromatography column. The insect cell supernatant medium was applied to a heparin-Toyopearl column as described under “Experimental Section”. The elution of protein from the affinity column was closely monitored by on-line UV detector at 280 nm (dark trace). The salt gradient is indicated by the gray line. The vertical lines indicate column fractions that were collected for further purification after measuring the enzymatic activity. B) Measurement of C5-epimerase activity based on radioactivity assay. Aliquots (20 μl) of indicated fractions were assayed for the C5-epimerase activity with [3H]-labeled N-sulfoheparosan polysaccharide as a substrate (1.5 × 105 CPM). After incubating with the enzyme for 24 hr, the sample was passed through a DEAE-Sepharose column (0.2 ml) to separate the N-sulfoheparosan polysaccharide from free 3H2O. The radioactivity of isolated polysaccharide was measured in the scintillation counter. C) DEEP-MS based assay to measure C5-epimerase activity during enzyme purification. The same fractions that were used in the radioactivity assay were utilized in the newly developed DEEP-MS assay for comparison. The aliquots of specific fractions were incubated with N-sulfoheparosan in D2O as described in the experimental section for 24 hr and then were treated with HNO2 at pH 1.5. The resultant glucuronyl/iduronyl-anhydromannose disaccharides were reduced with NaBH4 reagent, desalted on the mini Sephadex G10 column (1 cm ×10 cm) that was previously calibrated with known disaccharide standards and then analyzed by direct infusion into ESI-TOF-MS instrument to detect the molecular ions and their relative intensity. The relative activity of C5-epimerase was calculated based on the incorporation of deuterium into the substrate by measuring ion counts for DUA-AManR/HUA-AManR and plotted as a function of eluted fractions.