Volume 11, Issue 2 (Vol.11 No.2 Jul 2022)                   rbmb.net 2022, 11(2): 289-298 | Back to browse issues page


XML Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Novientri G, Sadikin M, Widia Jusman S. Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb). rbmb.net. 2022; 11 (2) :289-298
URL: http://rbmb.net/article-1-873-en.html
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.
Abstract:   (837 Views)
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.
Full-Text [PDF 324 kb]   (272 Downloads)    
Type of Article: Original Article | Subject: Biochemistry
Received: 2022/02/8 | Accepted: 2022/02/9 | Published: 2022/08/7

References
1. Geigenberger P, Fernie AR. Metabolic control of redox and redox control of metabolism in plants. Antioxid Redox Signal 2014; 21: 1389-1421. [DOI:10.1089/ars.2014.6018] [PMID] [PMCID]
2. Kang J, Pervaiz S. Mitochondria: Redox metabolism and dysfunction. Biochem Res Int 2012; 1: 1-14. [DOI:10.1155/2012/896751] [PMID] [PMCID]
3. Reeder B. Redox and Peroxidase Activities of the Hemoglobin Superfamily: Relevance to Health and Disease. Antioxidants Redox Signal 2017; 26: 763-776. [DOI:10.1089/ars.2016.6803] [PMID]
4. Reeder B, Svistunenko D, et al. The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology. Antioxid Redox Signal 2004; 6: 954-66. https://doi.org/10.1089/1523086042259832 [DOI:10.1089/ars.2004.6.954] [PMID]
5. Umbreit J. Methemoglobin - It's Not Just Blue : A Concise Review. Am. J. Hematol 2007; 144: 134-144. [DOI:10.1002/ajh.20738] [PMID]
6. Hultquist D, Passon P. Catalysis of methaemoglobin reduction by erythrocyte cytochrome b5 and cytochrome b5 reductase. Nat New Biol 1971; 229: 252-253. [DOI:10.1038/newbio229252a0] [PMID]
7. Faivre B, Menu P, Labrude P, et al. Hemoglobin Autooxidation/Oxidation Mechanisms and Methemoglobin Prevention or Reduction Processes in the Bloodstream Literature review and outline of autooxidation reaction. Artif Cell Blood Sub 1998; 26: 17-26. [DOI:10.3109/10731199809118943] [PMID]
8. Adler, Erich, Euler H, et al. Diaphorase I and II. Nature 1939; 143: 641. [DOI:10.1038/143641b0]
9. Huennekens F, Caffrey R, Basford R, et al. Erythrocyte metabolism: IV. Isolation and properties of methemoglobin reductase. J Biol Chem 1957; 227: 261-272. [DOI:10.1016/S0021-9258(18)70813-2]
10. Scott E. The relation of diaphorase of human erythrocytes to inheritance of methemoglobinemia. J Clin Invest 1960; 39: 1176-1179. [DOI:10.1172/JCI104131] [PMID] [PMCID]
11. Kitao T, Sugita Y, Yoneyama Y, et al. Methemoglobin reductase (cytochrome b5 reductase) deficiency in congenital methemoglobinemia. Blood 1974; 44: 879-884. [DOI:10.1182/blood.V44.6.879.879] [PMID]
12. Hagler L, Coppes Jr. RI, Herman RH. Identification dependent and enzyme. J Biol Chem 1979; 254: 6505-6514. [DOI:10.1016/S0021-9258(18)50397-5]
13. Arihara K, Greaser ML, Mozdziak PE. Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle. Meat Sci 1995; 39: 205-213. [DOI:10.1016/0309-1740(94)P1821-C]
14. Yoshizato K, Thuy LTT, Shiota G, et al. Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells. Proc Japan Acad Ser B Phys Biol Sci 2016; 92: 77-97. [DOI:10.2183/pjab.92.77] [PMID] [PMCID]
15. Sahara N, Sadikin M, Jusman S. The Enzyme that Reduces Oxidized Cytoglobin in Bovine Liver : An Exploration. Int. J. Pharma Med. Biol. Sci. 2020; 9: 129-133. [DOI:10.18178/ijpmbs.9.3.129-133]
16. Guo C, Gynn M, Chang T. Extraction of superoxide dismutase , catalase , and carbonic anhydrase from stroma- free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin - superoxide dismutase - catalase - carbonic anhydrase Extraction. Aritificial cells, Nanomedicine, Biotechnol 2015; 43: 157-162. [DOI:10.3109/21691401.2015.1035479] [PMID]
17. Murray, Robert K. Harper's Illustrated Biochemistry. New York: McGraw-Hill, 2003.
18. Mains I, Power D, Thomas E, et al. Diaphorase I (Di-1) [EC 1.6.99.-]. Biochem J 1980; 191: 457. [DOI:10.1042/bj1910457] [PMID] [PMCID]
19. Weller DL. Isoelectric point and molecular weight of a diaphorase of Entamoeba invadens. J Parasitol 1982; 68: 343-345. [DOI:10.2307/3281207] [PMID]
20. Kianmehr A, Mahdizadeh R, Oladnabi M, et al. Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus. 3 Biotech 2017; 7: 1-9. [DOI:10.1007/s13205-017-0763-0] [PMID] [PMCID]
21. Nguyen T, Phan KN, Lee J-B, Kim JG. Met-myoglobin formation, accumulation, degradation, and myoglobin oxygenation monitoring based on multiwavelength attenuance measurement in porcine meat. J Biomed Opt. 2016; 21(5):057002. [DOI:10.1117/1.JBO.21.5.057002] [PMID]
22. Zijlstra WG, Buursma A, Falke HE, Catsburg JF. Spectrophotometry of hemoglobin: absorption spectra of rat oxyhemoglobin , deoxyhemoglobin , carboxyhemoglobin , and methemoglobin. Comparative biochemistry and physiology b-Biochemistry & molecular biology 1994;107(I):161-6. [DOI:10.1016/0305-0491(94)90238-0]
23. Randi EB, Vervaet B, Tsachaki M, Porto E, Vermeylen S, Lindenmeyer MT, et al. The Antioxidative Role of Cytoglobin in Podocytes: Implications for a Role in Chronic Kidney Disease. Antioxid Redox Signal 2020; 32(16): 1155-1171. [DOI:10.1089/ars.2019.7868] [PMID]
24. Smagghe B, Hoy J, Percifield S, et al. Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins. Biopolymers 2009; 91: 1083-1096. [DOI:10.1002/bip.21256] [PMID]
25. Trent J, Watts R, Hargrove M. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J Biol Chem 2001; 276: 30106-30110. [DOI:10.1074/jbc.C100300200] [PMID]
26. Trent J, Hargrove M. A ubiquitously expressed human hexacoordinate hemoglobin. J Biol Chem 2002; 277: 19538-19545. [DOI:10.1074/jbc.M201934200] [PMID]
27. Dewilde S, Kiger L, Burmester T, et al. Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family. J Biol Chem 2001; 276: 38949-38955. [DOI:10.1074/jbc.M106438200] [PMID]
28. Smagghe B, Sarath G, Ross E, et al. Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species. Biochemistry 2006; 45: 561-570. [DOI:10.1021/bi051902l] [PMID]
29. Weiland T, Kundu S, Trent J, et al. Bis-histidyl hexacoordination in hemoglobins facilitates heme reduction kinetics. J Am Chem Soc 2004; 126: 11930-11935. [DOI:10.1021/ja046990w] [PMID]
30. Mathai C, Jourd'heuil FL, Lopez-Soler RI, et al. Emerging perspectives on cytoglobin, beyond NO dioxygenase and peroxidase. Redox Biol 2020; 32: 101468. [DOI:10.1016/j.redox.2020.101468] [PMID] [PMCID]
31. Kakar S, Hoffman FG, Storz JF, et al. Structure and reactivity of hexacoordinate hemoglobins. Biophys Chem 2010; 152: 1-14. [DOI:10.1016/j.bpc.2010.08.008] [PMID] [PMCID]

Add your comments about this article : Your username or Email:
CAPTCHA

Send email to the article author


Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2015 All Rights Reserved | Reports of Biochemistry and Molecular Biology

Designed & Developed by : Yektaweb