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Authors: Naxin Jiang , Nguan Soon Tan , Bow Ho & Jeak Ling Ding 


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 superoxide (1). 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


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.

Anticipated Results

The read out of CLA-LA varies from 10e5-10e7 rlu/s depending on the concentration of the metHb and the H2O2 as the substrate as well as that of CLA as the indicator.


  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).


Figure 1: The pseudoperoxidase activity of methemoglobin (metHb) was demonstrated by CLA-CL.

Fig 1

(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.

Associated Publications

Respiratory protein–generated reactive oxygen species as an antimicrobial strategy, Naxin Jiang, Nguan Soon Tan, Bow Ho, and Jeak Ling Ding, Nature Immunology 8 (10) 1114 - 1122 26/08/2007 doi:10.1038/ni1501

Author information

Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding, National University of Singapore

Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.485. Originally published online 31 October 2007.

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