Bortolami Silvia, Cavallini Lucia
Dipartimento di Chimica Biologica, Via G. Colombo 3, Università di Padova, Italy.
Free Radic Res. 2009 May;43(5):446-56. doi: 10.1080/10715760902870629.
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.
建立并验证了一种用于荧光检测瞬时表达催化剂辣根过氧化物酶（HRP）的细胞内过氧化氢的新方法。与HRP的更特异性反应极大地放大了所用氧化还原探针（2',7'-二氯二氢荧光素和二氢罗丹明）的氧化。HRP的表达不影响细胞活力。该方法揭示了完整转染细胞单层中作为主要细胞内过氧化氢来源的单胺氧化酶（MAO）活性。探针的氧化速率对MAO的激活/抑制有特异性反应。MAO产生的过氧化氢对其氧化对细胞内过氧化氢竞争剂敏感：当丙酮酸去除过氧化氢时其氧化速率降低，而当谷胱甘肽依赖性去除系统受损时其氧化速率增加。添加细胞外过氧化氢后也测量到了特异性反应。过氧化氢与内源性HRP反应后荧光探针的氧化克服了其用于细胞内过氧化氢检测时的大多数批评。该方法可用于酶标仪中的直接测定，并被提议用于检测生理病理细胞模型中过氧化氢的产生。
