2.3.3. DPP-IV enzyme equivalent activity

A dilution series of six concentrations of DPP-IV (from 0.12 U mL^-1 to 0.003 U mL^-1) in native state (non-bound) was prepared and 25 μL of each solution was transferred in triplicates to an Eppendorf tube together with 75 μL of 50 mM Tris-HCl buffer (pH 7.5) and 100 μL of a 0.2 mM solution of GPPN. The mixture was left to react for 30 minutes at 37°C under gentle shaking. Next, the solutions were transferred to a 96-well microplate; the absorbance of the samples was read at 405 nm and a calibration curve of the absorbances of the reaction product using DPP-IV in its native state was obtained. In parallel, 25 μL of a solution containing 0.05 mg of DPP-IV linked magnetic beads (theoretically equivalent to 10 mU of DPP-IV) was transferred to an Eppendorf tube in triplicate together with 75 μL of 50 mM Tris-HCl buffer (pH 7.5) and 100 μL of a 0.2 mM solution of GPPN. As mentioned above for non-bound DPP-IV, the mixture was left to react for 30 minutes at 37°C under gentle shaking. After the reaction time, the beads were magnetically separated, and the solution was transferred to a 96-well microplate and absorbance was read at 405 nm. The absorbance readings of the sample were interpolated in the calibration curve in order to calculate the equivalent activity of the immobilized DPP-IV in relation to DPP-IV in native state.