Biochemistry

Sebastian Mueller, MD, PhD

Professor of Medicine,

CAR, University of Heidelberg

VISCERA AG Bauchmedizin Bern

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Selected topics:

1. Detection of ultralow levels of hydrogen peroxide (H2O2)

2. H2O2 degradation in organelles and cells in real time

3. Enzymatic systems to mimic reactive oxygen species and hypoxia in cultured cells

References

1. Detection of ultralow levels of hydrogen peroxide (H2O2)

Fig. 1

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 ).

2. H₂O₂ degradation in organelles and cells in real time

Fig. 2

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.

3. Enzymatic model to mimic reactive oxygen species and hypoxia in cultured 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.

References

60. Exposing 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 Biol Med. 2013 Jul;60:325-35. doi: 10.1016/j.freeradbiomed. 2013.02.017. Epub 2013 Feb 26. PMID: 23485584 [PubMed - in process] Related citations

36. The GOX/CAT system: A novel enzymatic method to independently control hydrogen peroxide and hypoxia in cell culture.

Mueller S, Millonig G, Waite GN.

Adv Med Sci. 2009 Nov 27:1-15. [Epub ahead of print]PMID: 20022860 [PubMed - as supplied by publisher] Related articles

27. Hypoxia-inducible factor 1a under rapid enzymatic hypoxia: Cells sense decrements of oxygen but not hypoxia per se.

Millonig G, Heged�sch S, Becker L, Seitz HK, Schuppan D, Mueller S.

Free Radic Biol Med. 2009 Jan 15;46(2):182-191. Epub 2008 Nov 1.

PMID: 19007879 [PubMed - as supplied by publisher] Related Articles

18. Extracellular H2O2 and not superoxide determines the compartment-specific activation of transferrin receptor by iron regulatory protein 1.

Sureda A, Hebling U, Pons A, Mueller S.

Free Radic Res. 2005 Aug;39(8):817-24. PMID: 16036361 [PubMed - indexed for MEDLINE]

16. Myeloperoxidase-derived hypochlorous acid antagonizes the oxidative stress-mediated activation of iron regulatory protein 1.

M�tze S, Hebling U, Stremmel W, Wang J, Arnhold J, Pantopoulos K, Mueller S.

J Biol Chem. 2003 Oct 17;278(42):40542-9. Epub 2003 Jul 29. Erratum in: J Biol Chem. 2003 Dec 5;278(49):49662. PMID: 12888561 [PubMed - indexed for MEDLINE]

15. Sensitive and real-time determination of H2O2 release from intact peroxisomes.

Mueller S, Weber A, Fritz R, M�tze S, Rost D, Walczak H, V�lkl A, Stremmel W.

Biochem J. 2002 May 1;363(Pt 3):483-91. PMID: 11964148 [PubMed - indexed for MEDLINE]

10. Sensitive and nonenzymatic measurement of hydrogen peroxide in biological systems.

Mueller S.

Free Radic Biol Med. 2000 Sep 1;29(5):410-5. PMID: 11020662 [PubMed - indexed for MEDLINE]

7. Direct evidence for catalase as the predominant H2O2 -removing enzyme in human erythrocytes.

Mueller S, Riedel HD, Stremmel W.

Blood. 1997 Dec 15;90(12):4973-8. PMID: 9389716 [PubMed - indexed for MEDLINE]

5. Determination of catalase activity at physiological hydrogen peroxide concentrations.

Mueller S, Riedel HD, Stremmel W.

Anal Biochem. 1997 Feb 1;245(1):55-60. PMID: 9025968 [PubMed - indexed for MEDLINE]

4. Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils.

Mueller S, Arnhold J.

J Biolumin Chemilumin. 1995 Jul-Aug;10(4):229-37. PMID: 8533604 [PubMed - indexed for MEDLINE]

3. Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite.

Arnhold J, Mueller S, Arnold K, Sonntag K.

J Biolumin Chemilumin. 1993 Nov-Dec;8(6):307-13. PMID: 8285109 [PubMed - indexed for MEDLINE]

2. Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide.

Arnhold J, Mueller S, Arnold K, Grimm E.

J Biolumin Chemilumin. 1991 Jul-Sep;6(3):189-92. PMID: 1746319 [PubMed - indexed for MEDLINE]

1. On the action of hypochlorite on human serum albumin.

Arnhold J, Hammerschmidt S, Wagner M, Mueller S, Arnold K, Grimm E.

Biomed Biochim Acta. 1990;49(10):991-7. PMID: 1964376 [PubMed - indexed for MEDLINE]