Sebastian Mueller, MD, PhD
Professor of Medicine,
CAR, University of Heidelberg
VISCERA AG Bauchmedizin Bern
Hydrogen peroxide is present on our planet since the very beginning and it has played a critical role in the evolution of life. From 1990-1995, an ultrasensitive, non-enyzmatic assay (hypochlorite-luminol) was developed to detect hydrogen peroxide in biological fluids down to nanomolar concentrations. The figure shows the blue chemiluminescence derived from the oxidation of luminol by hypochlorite (ref. 4 reviewed in ref. 36). This assay has been the prerequisite for the fast enzyme kinetic studies below and the development of enzymatic model systems to study peroxide and hypoxia signaling in cells (see cell biology ).
Based on the above H2O2 assay, enzymatic kinetics of peroxide metabolizing enzymes such as catalase, glutathione peroxide or oxidases can be measured in real time without inactivating the enzymes. The above real-time measurement shows degradation of H2O2 by catalase (A), blocking of catalase by sodium azide (B) and demonstration of blocked catalase by addition of novel peroxide (C). See also ref. 5 and 7 . Valuable insights have been gained on how H2O2is removed e.g. by human erythrocytes or liver cells.
The above studies allowed to develop in the following years (first 1007) an enyzmatic system using glucose oxidase (GOX) and catalase (CAT) to independently control hydrogen peroxide and oxygen levels in cultured cells (reviewed e.g. in 36.). Such models are urgently needed to study the physiological functions of ROS in cultured cells. The stoichiometry of the GOX/CAT system is shown in Fig. 3. H2O2 is produced by GOX and consumed by CAT. Due to the specific kinetic properties of both enyzmes, stable H2O2 concentrations can be mainainted over hours (Fig. 4). (more details e.g. in 36.)
Fig. 3 Fig. 4
The GOX/CAT system been successfully explored in studying the communication between cells using as a singnaling molecule (see the Cell biology section). H2O2 levels are solely controlled by the ratio of GOX and CAT activities. They can be adjusted at non-toxic or toxic dosages over 24 hours. The additional hypoxia is controlled by the activity of the oxygen-consuming GOX and the diffusion distance of oxygen from the medium surface to the adhered cells (medium volume). In contrast to hypoxia chambers, the GOX/CAT system more rapidly induces hypoxia within minutes at a defined rate. Thus, the GOX/CAT system allows to adjust H2O2 levels under hypoxic conditions truly simulating H2O2 release e.g. by inflammatory cells or intracellular sources. GOX/CAT can be employed to address many questions ranging from redox signaling during inflammation to ischemia/reperfusion studies. More details are provided in the Cell biology section.
cells to H2O2: A quantitative comparison between continuous low-dose and
one-time high-dose treatments.
Sobotta MC, Barata AG, Schmidt U, Mueller S, Millonig G, Dick TP. Free Radic
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