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Published on: October 3, 2018
Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)
Gissi Novientri1, Mohamad Sadikin2,3, Sri Widia Jusman2,3
1Master Program of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
Insights
Bovine diaphorase, an enzyme that reduces methemoglobin (metHb), showed minimal activity in reducing metacytoglobin (metCygb). This suggests diaphorase is not a significant metCygb reductase in cells, unlike its known role with metHb.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Cytoglobin (Cygb) is an oxygen-binding protein involved in tissue oxygen transport.
- Oxidation of Cygb's heme iron to the ferric state (metCygb) results in loss of oxygen-binding capacity.
- Methemoglobin (metHb) and metmyoglobin (metMb) reduction is known, but metCygb reductase activity was unidentified.
Purpose of the Study:
- To investigate the potential of bovine diaphorase as a reductase for metacytoglobin (metCygb).
- To compare the reducing activity of diaphorase on metCygb versus its natural substrate, methemoglobin (metHb).
Main Methods:
- Purification of diaphorase from bovine erythrocytes using gel filtration and ion-exchange chromatography.
- Verification of enzyme purity via SDS-PAGE and Western Blot (WB).
- Spectroscopic determination of metCygb reduction by purified diaphorase.
Main Results:
- Purified bovine diaphorase exhibited a 10.77-fold increase in specific activity against metHb.
- The enzyme's Vmax for metCygb reduction was 13.7 times lower than that for metHb.
- Western blot confirmed enzyme purity using an anti-cyb5r3 antibody.
Conclusions:
- Bovine erythrocyte diaphorase demonstrates significantly lower reducing activity on metCygb compared to metHb.
- The purified diaphorase is not a potent enzymatic reductase for metCygb in vitro.
- Further research is needed to identify specific metCygb reductase enzymes.
Background:
Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe2+ ion in its heme group into metglobin (globin-Fe3+) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb.
Methods:
Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationic-exchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy.
Results:
The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin-1, which was 13.7 times less than the Vmax of metHb.
Discussion:
In conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.
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