COMPARATIVE-STUDY OF SOME SYNTHESIZED AND COMMERCIAL FLUOROGENIC SUBSTRATES FOR HORSERADISH-PEROXIDASE AND ITS MIMETIC ENZYME HEMIN BY A FLOW-INJECTION METHOD

Four 3,4-dihydroquinoxalin-2(1H)-one derivatives, i.e., 3,4-dihydroqui noxalin-2(1H)-one (DHQ), 3-methyl-3,4-dihydroquinoxalin-2(1H)-one (MDH Q), 3,4-dihydroquinoxalin-2(1H)-one-6-acid (DHQ-6-A) and 3-methyl-3,4- dihydroquinoxalin-2(1H)-one-6-acid (MDHQ-6-A), and N,N'-dicyanomethyl- o-phenylenediamine (DCM-OPA) were synthesised as potential substrates for horseradish peroxidase (HRP). Of these compounds DCM-OPA, DHQ and MDHQ can be prepared by very simple methods in a pure form in large qu antities. Their properties for use as fluorogenic substrates for HRP a nd its mimetic enzyme hemin were compared with commercially available substrates, i.e., p-hydroxyphenylacetic acid (p-HPA), p-hydroxyphenylp ropionic acid (p-HPPA), homovanillic acid (HVA) and tyramine, by a flo w injection method. The results showed that DCM-OPA and MDHQ were the best among the five synthesised substrates and p-HPPA and P-HPA are be tter than HVA and tyramine. Substrates p-HPPA, p-HPA, DCM-OPA and MDHQ showed comparable ability for H2O2 detection in HRP and hemin catalys ed reaction systems, with detection limits in the nmol l(-1) region. T he stability of DCM-OPA is better than MDHQ, but both are stable for a t least a month in a refrigerator.