The determination of kinetic constants
and IC50 values of inhibitors was carried out in parallel
to the continuous fluorescence assay by discontinuous assays analyzed
by means of reversed-phase high-performance liquid chromatography
(RP-HPLC). The reaction buffer, concentration of enzyme, substrates,
and inhibitors were carried out as described above. The reaction was
quenched by the addition of 0.5% acetic acid after 30 min of incubation
and centrifuged at 2000g at 37 °C for 15 min
to remove precipitated BSA and HDAC11. The reactions were analyzed
by RP-HPLC (Shimadzu, HPLC Prominence system) with a Kinetex 2.6 μm
XB-C18 100 Å column (100 × 3 mm; Phenomenex, Torrance, CA).
The mobile phase A was 5% acetonitrile with 0.1% (v/v) TFA and the
mobile phase B was 95% acetonitrile with 0.1% (v/v) TFA. The separation
of the reaction product from the acylated substrate was performed
in a 12-min linear gradient from 10 to 60% of eluent B at a flow rate
of 0.6 mL/min. The product and substrate peaks were quantified using
the absorbance at 365 nm (absorption of the 3-nitrotyrosyl moiety)
to verify the results of the fluorescence assay.
and IC50 values of inhibitors was carried out in parallel
to the continuous fluorescence assay by discontinuous assays analyzed
by means of reversed-phase high-performance liquid chromatography
(RP-HPLC). The reaction buffer, concentration of enzyme, substrates,
and inhibitors were carried out as described above. The reaction was
quenched by the addition of 0.5% acetic acid after 30 min of incubation
and centrifuged at 2000g at 37 °C for 15 min
to remove precipitated BSA and HDAC11. The reactions were analyzed
by RP-HPLC (Shimadzu, HPLC Prominence system) with a Kinetex 2.6 μm
XB-C18 100 Å column (100 × 3 mm; Phenomenex, Torrance, CA).
The mobile phase A was 5% acetonitrile with 0.1% (v/v) TFA and the
mobile phase B was 95% acetonitrile with 0.1% (v/v) TFA. The separation
of the reaction product from the acylated substrate was performed
in a 12-min linear gradient from 10 to 60% of eluent B at a flow rate
of 0.6 mL/min. The product and substrate peaks were quantified using
the absorbance at 365 nm (absorption of the 3-nitrotyrosyl moiety)
to verify the results of the fluorescence assay.