Detection of O2.- production by chemiluminescence (CL) assay
Lab/Group: Jeak Ling DING’s lab (National University of Singapore)
Related Journal & Article Information
Journal: Nature
Introduction
Hemoglobin is auto-oxidized from ferrous-hemoglobin to ferric hemoglobin (methemoglobin, metHb) with the production of H2O2. H2O2 initiates the catalytic cycle between the ferric (HbFeIII) and ferryl (HbFeIV) hemes, thus activating the pseudoperoxidase activity of metHb, which eliminates the H2O2, producing the superoxide1. The chemiluminescence (CL) of Cypridina luciferin analog indicates the generation of O2•- or singlet oxygen, but not that of ozone, hydroxyl radicals or H2O22. To further confirm that the ROS species produced by metHb and indicated by CLA-CL is superoxide anions but not singlet oxygen, superoxide dismutase (SOD) is applied as a diagnostic confirmation of superoxide anion production.
Materials
Reagents
Equipment
Procedure
The pseudoperoxidase activity is measured as described 2, with modifications.
1. Add metHb into a substrate mixture containing 5.3 μM of CLA and 3.3 mM of H2O2 in 150 μl of PBS (pH 7.4).
2. Continuously monitor the chemiluminescence, indicated by relative luminescence units (rlu), at one reading per s, for 1 min, with Sirius luminometer (Berthold Detection Systems GmbH, Germany).
3. The pseudoperoxidase activity of the metHb is designated as the relative luminescence units per second (rlu/s).
4. To prove that the ROS species indicated by CLA-CL is superoxide anions, 3 Units/ml of SOD is applied to specifically quench the superoxide anions.
Troubleshooting
Critical Steps
Anticipated Results
The read out of CLA-LA varies from 105-107 rlu/s depending on the concentration of the metHb and the H2O2 as the substrate as well as that of CLA as the indicator.
References
1. Alayash, A. I. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants? Nat Biotechnol. 17, 545-549 (1999).
2. Kawano, T., Pinontoan, R., Hosoya, H. & Muto, S. Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin. Biosci Biotechnol Biochem. 66, 1224-1232 (2002).
Acknowledgements
Keywords
Superoxide ion; chemiluminescene; Cypridina luciferin analog
Figure 1
The pseudoperoxidase activity of methemoglobin (metHb) was demonstrated by CLA-CL.
(a) metHb catalyzes the production of O2•-, as shown by the chemiluminescence (CL) assay. (b) SOD significantly reduces metHb-mediated CLA-luminescence, thereby confirming the superoxide anion.
