Enhanced detection of H2O2 in cells expressing horseradish peroxidase.
A new procedure for fluorescent detection of intracellular H2O2 in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2',7'-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H2O2 source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H2O2 is sensitive to intracellular H2O2 competitors: it decreases when H2O2 is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H2O2. Oxidation of the fluorescent probes following reaction of H2O2 with endogenous HRP overcomes most criticisms in their use for intracellular H2O2 detection. The method can be applied for direct determination in plate reader and is proposed to detect H2O2 generation in physio-pathological cell models.
Dipartimento di Chimica Biologica, Via G. Colombo 3, Università di Padova, Italy.
Silvia
S
Bortolami
Lucia
L
Cavallini
D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008995:Monoamine Oxidase / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D008995:Monoamine Oxidase