A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid.

Analytical biochemistry (2012-10-17)
Stéphane Mann, Luc Eveleigh, Olivier Lequin, Olivier Ploux
RESUMEN

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
2-Mercaptoethanol, ≥99.0%
Sigma-Aldrich
2-Mercaptoethanol, for molecular biology, suitable for electrophoresis, suitable for cell culture, BioReagent, 99% (GC/titration)
Sigma-Aldrich
Phthaldialdehyde, ≥97% (HPLC), powder (may contain lumps)
Sigma-Aldrich
2-Mercaptoethanol, BioUltra, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
Phthaldialdehyde Reagent, Solution Complete
Sigma-Aldrich
Phthaldialdehyde, suitable for HPLC fluorimetric detection of amino acids, ≥99% (HPLC), powder
Sigma-Aldrich
Phthaldialdehyde, for fluorescence, ≥99.0% (HPLC)
Sigma-Aldrich
Phthaldialdehyde Reagent, Solution Incomplete