2.10 Recombinant protein inhibition assay in vitro
The VIN activity of recombinant PpVIN2 was assayed as described in Section 2.8, and expressed as U/mg protein. Inhibitory effects were determined at different pH levels and temperatures by measuring the VIN activity of PpVIN2 in the presence of PpINH1. PpVIN2 plus double distilled water served as control. The inhibition rate was calculated by the following formula: Inhibition rate = [(PpVIN2+ddH2O) – (PpVIN2+ PpINH1)]/ (PpVIN2+ddH2O), where each term refers to VIN activity measured in the presence (+PpINH1) or absence (+ddH2O) of inhibitor.
The mixture was incubated at 37 °C for 30 minutes in assay buffers at pH 3.2, 3.6, 4.0, 4.4, 4.8, 5.2, 5.6, and 6.0. Sucrose was then added and enzyme activity was measured. These reactions were repeated at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, and 55 °C.