Kinetic properties and heme pocket structure of two domains of the polymeric hemoglobin of Artemia in comparison with the native molecule
Borhani, H.; Berghmans, H.; Trashin, S.; De Wael, K.; Fago, A.; Moens, L.; Habibi-Rezaei, M.; Dewilde, S. (2015). Kinetic properties and heme pocket structure of two domains of the polymeric hemoglobin of Artemia in comparison with the native molecule. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1854(10): 1307-1316. https://dx.doi.org/10.1016/j.bbapap.2015.05.007
In: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. Elsevier: Amsterdam. ISSN 1878-1454, more
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 Anemia, 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 O-2 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 P-50 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 -0.13 V versus the standard hydrogen electrode was determined for AfHb by direct electrochemical measurements. It is about 0.06 V higher than the potential of the single domain AsHbC1D5. This work shows that each domain in the hemoglobin of Anemia has different characteristics of ligand binding.
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