A HIGHLY SENSITIVE FLUORESCENT MICROASSAY OF H2O2 RELEASE FROM ACTIVATED HUMAN-LEUKOCYTES USING A DIHYDROXYPHENOXAZINE DERIVATIVE

This study describes a simple, reliable, highly sensitive and quantita tive fluorescence microplate-assay of H2O2 from activated leukocytes u sing a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihy droxyphenoxazine (A6550). Unlike the widely used fluorescent HRP subst rate scopoletin, A6550 is non-fluorescent and becomes highly fluoresce nt upon HRP-catalyzed H2O2 oxidation. Using 50 mu M A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-ace tate-activated human eosinophils and neutrophils is found to have a li near cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respect ively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as lon g as the ratio of A6550:H2O2 in the reaction mixture is higher than fi ve. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. W hen the concentrations of A6550 and HRP were 10 mu M and 0.2 U/ml, res pectively, as low as 2 pmol of H2O2 could be reliably measured. The se nsitivity of A6550/H2O2 assay is found to be at least 10-fold higher t han with scopoletin as the HRP substrate, The protocol described in th is study using A6550 to measure H2O2 release from activated granulocyt es can be easily adapted to other cell types which generate H2O2.