3.2.3. Enzymatic Assays with PTP1B

PTP1B activity was determined as follow. An aliquot of human recombinant PTP1B was diluted in the assay buffer containing 0.075 M β-β-dimethylglutarate pH 7.0, 1 mM EDTA, 0.1 mM DTT and p-nitrophenyl phosphate (pNPP) as substrate. After an appropriate interval time, the reaction was stopped diluting assay solution with 2 mL of KOH 0.1 M. The amount of p-nitrophenol released was determined measuring the absorbance of the solution at 400 nm using a spectrophotometer and a 1-cm optical pathlength (ε_mM of p-nitrophenol is 18).

The IC₅₀ value was determined measuring the pNPP hydrolysis rate in the presence of increasing inhibitor concentration. For each inhibitor, 15–16 different concentrations were used. All tests were carried out in triplicate and data obtained were normalized respect to control sample. Experimental points were fitted using Equation (1), shows in the Section 3.2.2.