| Assay Method Information | |
| | HPP Tautomerase Assay |
| Description: | Inhibition of the tautomerase activity of MIF was measured using the substrate 4-hydroxyphenyl pyruvic acid (HPP) in a procedure largely adapted from previous reports. A solution of HPP (10 mM) in acetate buffer (0.5 M ammonium acetate, pH adjusted to 6.0) was prepared and allowed to incubate overnight in the dark at room temperature to allow for equilibration of the keto and enol forms. Following the incubation period, the HPP solution was stored at 4 C. and used for no more than a week. For Ki determination, MIF protein (final concentration: ca. 50 nm) and inhibitor (multiple concentrations in DMSO, maintaining a final DMSO concentration of 1%) were incubated in borate buffer (0.5 M boric acid, pH 6.2) in a U-bottom 96-well plate (Falcon) for 20 minutes. A negative control (containing water and DMSO in lieu of protein and inhibitor, respectively) and a positive control (containing DMSO in lieu of inhibitor) were also prepared. The reaction began upon the addition of HPP solution (final concentration: 1.0 mM and 2.5 mM). The absorbance was monitored at 305 nm for the formation of the borate-enol complex using a Tecan INFINITE F500 plate reader over 175 seconds. Absorbance was measured three times for each [inhibitor]-[HPP] combination. Calculation of initial velocities and the nonlinear regression analyses for the enzyme kinetics were performed using the program Prism 6 (GraphPad), setting the Michaelis-Menten constant (Km) to 2.4. Reported Ki values represent the average value obtained from two assays performed on different days. (R)-ISO-1 (purchased from Santa Cruz Biotechnology) was used as a control. |
| Affinity data for this assay | |
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