Abstract |
Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).
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Authors | J Brian Jameson 2nd, Victor Kenyon, Theodore R Holman |
Journal | Analytical biochemistry
(Anal Biochem)
Vol. 476
Pg. 45-50
(May 01 2015)
ISSN: 1096-0309 [Electronic] United States |
PMID | 25712042
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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Copyright | Copyright © 2015 Elsevier Inc. All rights reserved. |
Chemical References |
- Linoleic Acids
- Lipoxygenase Inhibitors
- 13-hydroxy-9,11-octadecadienoic acid
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Topics |
- Chromatography, High Pressure Liquid
- Humans
- Linoleic Acids
(chemistry)
- Lipoxygenase Inhibitors
(chemistry)
- Mass Spectrometry
(methods)
- Molecular Structure
- Substrate Specificity
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