title: Kinetic properties and heme pocket structure of two domains of the polymeric hemoglobin of Artemia in comparison with the native molecule.
abstract: In this project, we studied some physicochemical properties of two different globin domains of the polymeric hemoglobin of the brine shrimp Artemia salina and compared them with those of the native molecule. Two domains (AsHbC1D1 and AsHbC1D5) were cloned and expressed in BL21(DE3)pLysS strain of Escherichia coli. The recombinant proteins as well as the native hemoglobin (AfHb) were purified from bacteria and frozen Artemia, respectively by standard chromatographic methods and assessed by SDS-PAGE. The heme environment of these proteins was studied by optical spectroscopy and ligand-binding kinetics (e.g. CO association and O2 binding affinity) were measured for the two recombinant proteins and the native hemoglobin. This indicates that the CO association rate for AsHbC1D1 is higher than that of AsHbC1D5 and AfHb, while the calculated P50 value for AsHbC1D1 is lower than that of AsHbC1D5 and AfHb. The geminate and bimolecular rebinding parameters indicate a significant difference between both domains. Moreover, EPR results showed that the heme pocket in AfHb is in a more closed conformation than the heme pocket in myoglobin. Finally, the reduction potential of -0.13V versus the standard hydrogen electrode was determined for AfHb by direct electrochemical measurements. It is about 0.06V higher than the potential of the single domain AsHbC1D5. This work shows that each domain in the hemoglobin of Artemia has different characteristics of ligand binding.
authors.affiliation: School of Biology, College of Science, University of Tehran, Tehran, Iran; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address: heshmat.akbariborhani@uantwerpen.be.
authors.forename: Heshmat Akbari
authors.initials: HA
authors.lastname: Borhani
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address: herald.berghmans@uantwerpen.be.
authors.forename: Herald
authors.initials: H
authors.lastname: Berghmans
authors.affiliation: Department of Chemistry, University of Antwerp, Antwerp, Belgium. Electronic address: stanislav.trashin@uanwerpen.be.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Trashin
authors.affiliation: Department of Chemistry, University of Antwerp, Antwerp, Belgium. Electronic address: karolien.dewael@uantwerpen.be.
authors.forename: Karolien
authors.initials: K
authors.lastname: De Wael
authors.affiliation: Department of Bioscience, Zoophysiology, Aarhus University, Aarhus, Denmark. Electronic address: angela.fago@biology.au.dk.
authors.forename: Angela
authors.initials: A
authors.lastname: Fago
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address: luc.moens@uantwerpen.be.
authors.forename: Luc
authors.initials: L
authors.lastname: Moens
authors.affiliation: School of Biology, College of Science, University of Tehran, Tehran, Iran; Nano-Biomedicine Center of Excellence, Nanoscience and Nanotechnology, Research Center, University of Tehran, Tehran, Iran. Electronic address: mhabibi@ut.ac.ir.
authors.forename: Mehran
authors.initials: M
authors.lastname: Habibi-Rezaei
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address: sylvia.dewilde@uantwerpen.be.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Dewilde
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000737:chemistry* / Q000378:metabolism; D060829:Arthropod Proteins / Q000737:chemistry* / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D058105:Polymerization; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D060829:Arthropod Proteins; D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen
keywords: Artemia hemoglobin; Heme pocket structure; Ligand binding kinetics; Redox potential