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en Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb) بیوشیمی Biochemistry مقالات اصلی Original Article <div style="text-align: justify;">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 cationicexchanger 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. Conclusions: In conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.</div> Bovine Erythrocyte, Cytochrome B5 Reductase, Diaphorase, Metcytoglobin, Reduction. 289 298 http://rbmb.net/browse.php?a_code=A-10-946-1&slc_lang=en&sid=1 Gissi Novientri 100319475328460012989 100319475328460012989 No Master Program of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. Mohamad Sadikin sadikinmohamad@gmail.com. 100319475328460012990 100319475328460012990 Yes Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia & Center of Hypoxia and Oxidative Stress Studies, Faculty of Medicine, Universitas Indonesia. Sri Widia Jusman 100319475328460012991 100319475328460012991 No Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia & Center of Hypoxia and Oxidative Stress Studies, Faculty of Medicine, Universitas Indonesia