Arginase Inhibitor Screening Service

Arginase Activity Assay Control Tests: Km for Arginase I and IC50 Determination for ABH

Summary
In order to test the suitability of BioAssay Systems Arginase Assay for screening for arginase inhibitors, we performed some initial experiments to determine the Km of L-arginine for a purified human arginase and the IC50 for a known arginase inhibitor, ABH. We measured a Km of 3.4 ± 0.2 mM. Using 3 mM L-arginine, we observed an IC50 = 0.14 ± 0.1 µM for ABH. We also verified that 2 v% DMSO will not affect the assay.

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Results
Km of L-Arginine
In order to determine the Km of L-arginine for Arginase I, we titrated L-arginine from 0–20 mM with 25 ng Arginase I and computed the rates of reaction, V (µmol/min/mg), for each L-arginine concentration. The rates of reaction were then plotted versus L-arginine and the Km was computed using Prism 4 (GraphPad Software Inc). We found the Km = 3.4 ± 0.2 mM.

arginase inhibitor figure1

ABH IC50 Determination
The IC50 determination required three steps: 1) 30 min pre-incubation of 25 ng arginase I with ABH, 2) arginase reaction with 3 mM L-arginine for 25 min and 3) urea concentration measurement. We titrated ABH from 0-10 µM and computed the rate of reaction, V, for each ABH concentration. The rates of reaction were then plotted versus the Log [ABH] and the IC50 was computed using Prism 4 (GraphPad Software Inc). We observed an IC50 = 0.14 ± 0.01 µM.

arginase inhibitor figure2

Effect of DMSO
In order to determine if DMSO would interfere with this arginase assay, we set up four reactions: 25 ng arginase I ± 2 v% DMSO and 0 ng arginase ± 2 v% DMSO. Following a 20 min reaction and addition of the Urea Reagent, we measured the OD520 and OD430 and computed the ΔOD (ODarginase – ODno arginase) for the 2 v% DMSO and 0 v% DMSO reactions at each wavelength. The following table displays the results:

Sample ΔOD520 ΔOD430
2 v% DMSO 0.3995 1.4476
0 v% DMSO 0.3978 1.4181

Clearly 2 v% DMSO has no adverse effects on the assay and will not be an issue for the compound screen.

Conclusions
The results for the Km of L-arginine for arginase I and IC50 of ABH observed in this study agree well with numbers found in literature (within 3 fold). This study also verified that 2 v% DMSO will not interfere with the assay. Thus, this arginase assay will be suitable for screening for arginase inhibitors.


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