Abstract
Recently, we have described a method for evaluation of plasma amine oxidase (PAO) inhibitors, which monitors the formation of 6-(5-phenyl-2H-tetrazol-2-yl)hexanal from the corresponding amine substrate by HPLC with UV-detection using purified bovine PAO. We now investigated, whether crude bovine plasma can be used as enzyme source in this assay instead of the purified enzyme. With the aid of specific inhibitors, it was ensured that there was no detectable activity of other important amine oxidases in the plasma, namely monoamine oxidase (MAO) A and B and diamine oxidase (DAO). For a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkan-1-amine substrates similar conversion rates were measured for both the purified PAO and crude plasma. The inhibition values determined for the PAO inhibitor 2-(4-phenylphenyl)acetohydrazide (16) under different conditions also corresponded. Additionally, inhibition data of the known PAO inhibitor 2-amino-N-(3-phenylbenzyl)acetamide (17) and a newly synthesised meta-substituted derivative of 16 were determined, which together reflect the two-step inhibition mechanism of these covalent inhibitors.
Highlights
Plasma amine oxidase (PAO) is a topaquinone- and copperdependent oxidase, which converts primary amines to aldehydes with the concomitant production of ammonia and hydrogen peroxide[1]
The enzyme, which is known as copper-containing amine oxidase 3 (AOC3), semicarbazide-sensitive amine oxidase (SSAO) or vascular adhesion protein-1 (VAP-1), is mainly expressed in endothelial cells of blood vessels, smooth muscle cells, and adipocytes
Since the supplier of the enzyme could not deliver the product for some time, we studied, whether bovine plasma itself can be used for inhibitor screening instead of the purified enzyme
Summary
Plasma amine oxidase (PAO) is a topaquinone- and copperdependent oxidase, which converts primary amines to aldehydes with the concomitant production of ammonia and hydrogen peroxide[1]. The conversion rates of the amine substrates 1–7 to the corresponding aldehydes 8–14 by bovine plasma was measured as described previously[33], with the modification that the solution of the isolated PAO in PBS (0.19 mg/mL) Determination of the IC50-values of the PAO inhibitor 2-(4-phenylphenyl)acetohydrazide (16) with bovine plasma using different substrates.
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