A spectrophotometric assay for monoamine oxidase activity with 2, 4-dinitrophenylhydrazine as a derivatized reagent.
A simple, rapid and reliable spectrophotometry was developed to determine monoamine oxidase (MAO). In this study, 2,4-dinitrophenylhydrazine (DNPH), a classic derivatizing reagent, was used to detect MAO-dependent aldehyde production; and traditional DNPH spectrophotometry was simplified. Benzylamine and serotonin oxidation were catalyzed by MAO-B and MAO-A, respectively, to aldehydes. These were derivatized with DNPH, and the corresponding quinones were further formed by adding NaOH. These DNPH derivatives with large conjugated structures were directly measured spectrophotometrically at 465 nm and 425 nm, without the need for precipitating, washing and suspending procedures. The addition of NaOH caused a red shift of the maximum absorption wavelength of these derivatives, which reduced the interference of free DNPH. MAO-B protein was as low as 47.5 μg in rat liver with correlation coefficients ranging within 0.995-0.999. This method is 2-3 times more sensitive than direct spectrophotometry. The detection of MAO inhibition through this method showed that IC50 values of rasagiline are 8.00 × 10(-9) M for MAO-B and 2.59 × 10(-7) M for MAO-A. These results are similar to the values obtained by direct spectrophotometry. Our study suggests that DNPH spectrophotometry is suitable to detect MAO activity, and has the potential for MAO inhibitor screening in the treatment of MAO-mediated diseases.
['Huang|Guili|G|', 'Zhu|Fei|F|', 'Chen|Yuhang|Y|', 'Chen|Shiqiang|S|', 'Liu|Zhonghong|Z|', 'Li|Xin|X|', 'Gan|Linlin|L|', 'Zhang|Li|L|', 'Yu|Yu|Y|']
D000818:Animals D004353:Drug Evaluation, Preclinical D008099:Liver D008297:Male D008995:Monoamine Oxidase D008996:Monoamine Oxidase Inhibitors D010659:Phenylhydrazines D051381:Rats D017207:Rats, Sprague-Dawley D013053:Spectrophotometry
Monoamine oxidase 2,4-dinitrophenylhydrazine DNPH spectrophotometry