Gissi Novientri - Master Program of Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
Mohamad Sadikin - 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.

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 Fe۲+ ion in its heme group into metglobin (globin-Fe۳+) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb۵r۳. 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=۳۰.۰۹ kDa) was purified ۱۰.۷۷-fold from crude enzyme with specific activity against metHb ۸.۴۷۹ U/mg. The purity was confirmed by WB using primary antibody anti-cyb۵r۳. The purified enzyme reduced metCygb at ۰.۷۸۵ μgmin-۱, which was ۱۳.۷ 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.

Bovine Erythrocyte, Cytochrome B۵ Reductase, Diaphorase, Metcytoglobin, Reduction.