Assay of N1-Acetylpolyamine Oxidase Activity with N1,N11-Didansylnorspermine as the Substrate by Ion-Pair Reversed Phase High Performance Liquid Chromatography

Abstract

An assay method to measure N 1 -acetylpolyamine oxidase (PAO) activity with N 1 ,N 11 -didansylnorspermine (DiDNS333) as the substrate by high performance liquid chromatography (HPLC) was developed. Dansylpolyamines were synthesized, and their activity as a substrate for partially purified PAO from rat liver was evaluated. Among the dansylpolyamines, DiDNS333 was a useful substrate for the development of the PAO assay method. DiDNS333 was degraded by PAO to 1-dansylamido-3-propanal (DNS3al) and N 1 -dansylnorspermidine (DNS33). As DNS3al was separated into two peaks by HPLC of the assay mixture containing aminoguanidine, determination of DNS33 was used for the development of the assay method. When the assay method was applied to inhibition studies, the DNS33 peak on the chromatogram was consistently produced, and weak interference was found in the incubation with higher concentrations of natural polyamines. This result suggested that contaminating polyamines in biological samples do not interfere with this method. When the assay method was applied to cell extract from Chinese hamster ovary cell samples, the PAO activity, even at the low level in the cells, could easily be detected with as little as 10 mg of protein, which corresponds to 1؋10 5 cells. This HPLC method is a rapid, sensitive and useful assay for the measurement of PAO activity.