Cuvette-Based Assay for Inhibitors of 12-hLO (12-human lipoxygenase): 8HQ Series - Round 1
The enzyme activity of 12-hLO was determined by a direct measurement of product formation by monitoring the absorbance at 234 nm in a 2 mL cuvette. IC50 values of inhibitors were obtained by measuring the enzymatic rate at a variety of concentrations (Carroll et al, Segraves et al). ..more
BioActive Compounds: 21
Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, Christopher P.
Screening Center: NIH Chemical Genomics Center
The enzyme activity of 12-hLO was determined by a direct measurement of product formation by monitoring the absorbance at 234 nm in a 2 mL cuvette. IC50 values of inhibitors were obtained by measuring the enzymatic rate at a variety of concentrations (Carroll et al, Segraves et al).
For control experiments, 2 mL of substrate buffer (10 uM arachidonic acid / 25 mM HEPES / 0.01% (v/v) Triton X-100, pH 8.0) was aliquoted in a cuvette with a magnetic stir bar. After equilibrium was ensured, an aliquot of inhibitor solvent was added (DMSO), and equilibrium was once again assured. The reaction was started by adding enzyme to the cuvette and the reaction was followed until completed. The inhibition experiments were performed as above, except the actual inhibitory compound was added instead of vehicle. To achieve an IC50, typically 5 concentrations of the inhibitor were studied. If the inhibitor concentration was constant, then five different reaction volumes were used.
Step \ Parameter \ Value \ Description
1; UV-Vis; 234 nm; Equilibration
2; Reagent; 2 mL; Substrate buffer
3; UV-Vis; 234 nm; Equilibration
4; Reagent; varies; Compound
5; UV-Vis; 234 nm; Equilibration
6; Reagent; 50-200 nM; Enzyme 12-hLO
7; UV-Vis; 234 nm; Enzymatic reaction
Step \ Notes
1; Blank cuvette to air.
2; 10 uM AA, 25 mM HEPES, 0.01% Triton X-100, pH 8.
3; No change in absorbance ensured.
4; Compound vehicle or compound in various concentrations depending on potency.
5; No change in absorbance ensured.
6; Enzyme concentration to achieve reaction rate between 0.02 and 0.04 AU/s.
7; Enzymatic reaction visualized until completion.
Keywords: NIH Roadmap, MLSCN, MLPCN, MLI, MLSMR, qHTS, NCGC, human
lipoxygenase, 12hLO, 12-hLO
For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given based on -Log[Potency*10^-6]
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)