Analytical centrifugation

The sedimentation velocity and sedimentation equilibrium of each NylC enzyme (0.34 mg ml⁻¹) in phosphate buffer were examined at 20 °C using analytical ultracentrifugation. Centrifugation was performed at 40,000 rpm and 14,000 rpm for sedimentation velocity and equilibrium experiments, respectively, by monitoring the absorbance at 280 nm at intervals of 5 min. Analytical ultracentrifugation experiments were performed using a Beckman-Coulter Optima XL-A analytical ultracentrifuge (Fullerton, CA) after pre-centrifugation at 3,000 rpm for 5 min. Double-sector 12-mm-thick charcoal-epon centerpieces and matched quartz windows were used for all experiments. The experimental sedimentation coefficients were corrected to s20, w, the sedimentation coefficient expressed in terms of the standard state of water at 20 °C, with the van Holde-Weischet method using the software UltraScan 8.0 (www.ultrascan.uthscsa.edu). Frictional ratios and partial specific volumes were also obtained using the UltraScan software³¹.