A Study Of A Novel Fluorogenic Probe For Monoamine Oxidase

Monoamine oxidases(MAO,EC 1.4.3.4),which are integral proteins located in the mitochondrial outer membrane,are flavin adenosine dinucleotide enzymes.In mammals,MAOs have two isoenzymes MAOA and MAOB which differ in specificity for substrates,sensitivity to inhibitors and primary amino acid sequence.In general,MAOs have large influence on neuropathic diseases such as Parkinson's syndrome, Alzheimer's disease,and other unhealthy behavior including smoking, alcoholism,aggressive behavior even the growth inhibition and progression of cancer.Therefore,MAOs have received an extensive attention on developing new drugs as targets.Although many methods,such as spectrophotometric,radiometric,HPLC and fluorometric assays are available to detect the activity of MAO,most of them are not ideal for high-throughput screening.Due to extraordinary sensitivity,good selectivity,short delay time(＜10^-9 s) and noninvasivity,fluorescent probe is an important analytical tool that has been widely used in pharmaceutical and biology.This paper desigened a novel fluorescent probe:4-methyl-7-(3-aminopropoxy) coumarin for MAO.This probe is based on the reaction mechanism proposed in the reference.With the treatment of MAO, 3-aminopropoxy is oxidative deaminated to form an aldehyde product and strongly fluorescent coumarin was generated.By comparing the intensity of the fluorescence before and after the reaction,the activities of MAOs could be easily detected.Herein we fisrtly successfully synthesized the probe,Second,we respectively purified the MAOA,B from human placenta and beef liver. Thirdly,the conditions of assays were optimized and the best condition was 2 h,37℃,borate buffer(pH7.4).Last,the constants of MAOs were determined:MAOA:K_m=16.04±0.05μmol/L,V_max=0.72±0.01 nmol/min/mg;MAOB:K_m=12.25±0.05 umol/L,V_max=2.57±0.02 nmol/min/mg.Simultaneity the appilications were studied including purification of the MAOs and the serums of different patients.In conclusion,[4-methyl-7-(3-aminopropoxy) coumarin]is simple, effective and sensitive probe for MAOA and B.