caa a22 4500 47574456X CHVBK 20180406123509.0 cr unu---uuuuu 170329e20000201xx s 000 0 eng 10.1023/A:1007086717412 doi (NATIONALLICENCE)springer-10.1023/A:1007086717412 Subunit Distribution of Calcium-Binding Sites in Lumbricus Terrestris Hemoglobin [Elektronische Daten] [Askar Kuchumov, Joseph Loo, Serge Vinogradov] The giant, ∼3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36-42 linkers L1-L4 (24-32 kDa). It is known to contain 50-80 Ca and 2-4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH ∼2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca2+ and all the Cu and Zn, the globin subassembly had 0.4 to ∼3 Ca2+, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH ∼2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH ∼2.2 and at neutral pH demonstrated avid binding of 1 Ca2+ and additional weaker binding of 7 Ca2+ in the presence of added Ca2+. Based on these and previous results which document the heterogeneous nature of the Ca2+-binding sites in Lumbricus Hb, we propose three classes of Ca2+-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) ∼30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) ∼25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1-100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding. Plenum Publishing Corporation, 2000 Lumbricus terrestris nationallicence hemoglobin nationallicence calcium content nationallicence calcium-binding sites nationallicence Kuchumov Askar Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 48201, Detroit, Michigan aut Loo Joseph Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 48105, Ann Arbor, Michigan aut Vinogradov Serge Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 48201, Detroit, Michigan aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/2(2000-02-01), 139-149 0277-8033 19:2<139 2000 19 10930 https://doi.org/10.1023/A:1007086717412 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1007086717412 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kuchumov Askar Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 48201, Detroit, Michigan aut NATIONALLICENCE 700 1- Loo Joseph Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 48105, Ann Arbor, Michigan aut NATIONALLICENCE 700 1- Vinogradov Serge Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 48201, Detroit, Michigan aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/2(2000-02-01), 139-149 0277-8033 19:2<139 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer