Spectramax plus

PpEst was pre-incubated for 1 h in buffers ranging from pH 4 to 11 (pH 4–6: 0.1 M citric acid buffer, pH 7–9: 0.1 M Tris-HCl, pH 10–11: 0.1 M borate buffer). 4-NPB was added to the assay buffer (150 mM NaCl, 0.01% Triton X-100, 20 mM buffer with appropriate pH) to a final concentration of 0.1 mM and the reaction initiated by adding 1 μg enzyme in 7.5 μL to wells in a 96 well plate. Production of 4-nitrophenolate and 4-nitrophenol was measured at 348 nm (their isosbestic point) (Biggs 1954 ) continuously for 10 min at 30 °C on a Spectramax Plus (BMG-LABTECH, Germany). Autohydrolysis of 4-NPB was determined by addition of 7.5 μL of the corresponding buffer without enzyme.

One hundred nanograms of PpEst were incubated in 100 μL 100 μM 4-NPB in assay buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton –X 100) in a 96 well plate together with BuTA in concentrations ranging from 0 to 1 mM. Activity was determined by the appearance of 4-nitrophenolate at 405 nm on a Spectramax Plus (BMG-LABTECH, Germany) and is given as activity relative to rate when no BuTA was present. K_I was determined from the function derived using the Sigmaplot 13.0 (Systat Software Inc., Germany) regression wizard equation ‘Ligand binding; one site competition’.

Assay buffer of 150 mM NaCl, 0.01% Triton X-100 and 20 mM Tris-HCl pH 8.0 was heated to temperatures between 25 and 90 °C in 5-degree increments. One microgram of PpEst in 7.5 μL 100 mM Tris-HCl was incubated with 1 mL of the buffer solution for 1 min before 4-NPB was added to a final concentration of 0.1 mM. Production of 4-nitrophenolate was measured continuously at 405 nm for 3 min on a Spectramax Plus (BMG-LABTECH, Germany). Autohydrolysis of 4-NPB was determined by the addition of 7.5 μL 100 mM Tris-HCl (pH 7.0) without enzyme.