Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

Gissi Novientri; Mohamad Sadikin; Sri Widia Jusman

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

Gissi Novientri, Mohamad Sadikin, Sri Widia Jusman

Department of Biochemistry and Molecular Biology Pre Clinic

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	289-298
Number of pages	10
Journal	Reports of Biochemistry and Molecular Biology
Volume	11
Issue number	2
Publication status	Published - Jul 2022

Keywords

Bovine erythrocyte
Cytochrome b5 reductase
Diaphorase
Metcytoglobin
Reduction

Cite this

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title = "Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)",

abstract = "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.",

keywords = "Bovine erythrocyte, Cytochrome b5 reductase, Diaphorase, Metcytoglobin, Reduction",

author = "Gissi Novientri and Mohamad Sadikin and Jusman, {Sri Widia}",

note = "Funding Information: This research was financially supported by the PUTI grant from Universitas Indonesia with contract number NKB- 1837/UN2.RST/HKP.05.00/2020. Publisher Copyright: {\textcopyright} 2022. Reports of Biochemistry & Molecular Biology.All Rights Reserved.",

year = "2022",

month = jul,

language = "English",

volume = "11",

pages = "289--298",

journal = "Reports of Biochemistry and Molecular Biology",

issn = "2322-3480",

publisher = "Varastegan Institute for Medical Sciences",

number = "2",

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TY - JOUR

T1 - Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

AU - Novientri, Gissi

AU - Sadikin, Mohamad

AU - Jusman, Sri Widia

N1 - Funding Information: This research was financially supported by the PUTI grant from Universitas Indonesia with contract number NKB- 1837/UN2.RST/HKP.05.00/2020. Publisher Copyright: © 2022. Reports of Biochemistry & Molecular Biology.All Rights Reserved.

PY - 2022/7

Y1 - 2022/7

N2 - 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.

AB - 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.

KW - Bovine erythrocyte

KW - Cytochrome b5 reductase

KW - Diaphorase

KW - Metcytoglobin

KW - Reduction

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SN - 2322-3480

VL - 11

SP - 289

EP - 298

JO - Reports of Biochemistry and Molecular Biology

JF - Reports of Biochemistry and Molecular Biology

IS - 2

ER -

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

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