Figure 2

Assay development for rhCHST4. The enzyme was assayed with the acceptor substrate of a disaccharide GlcNAcMan. A) Activity-enzyme plot and the linear response region. The reactions were performed with fixed substrate inputs (2,500 pmol GlcNAcMan and 1,000 pmol of PAPS) but varied enzyme input from 0 to 500 ng. The calculated activity was then plotted against the enzyme inputs. The linear response region was located from 0 to 40 ng of enzyme input. Last lane contained no acceptor substrate. B) The experiment was repeated in the linear region. A linear least-square line was fitted to the activity-enzyme plot and the slope of the line was the specific activity for rhCHST4. The second lane contained no acceptor substrate. C) A product-time course of rhCHST4. All reactions contained 7.5 ng enzymes but reaction time varied from 0 to 20 minutes. The relative mobilities of the product and PAPS were slightly different due to variations on the electrophoresis buffer preparation. The faint spots right above the PAPS were likely due to APS, a PAPS degradation product, which was observed especially with aged PAPS preparation.