Antagonistic interaction between oxygenation-linked lactate and CO 2 binding to human hemoglobin Mette Søby Nielsen, Roy E. Weber ⁎ Zoophysiology, Department of Biological Sciences, University of Aarhus, DK8000 Aarhus, Denmark Received 4 October 2006; received in revised form 1 December 2006; accepted 1 December 2006 Available online 12 December 2006 Abstract Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO 2 , lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de) oxygenation. We analysed the effects of lactate and CO 2 , separately and in combination, on O 2 binding of purified human Hb at 37 °C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO 2 binding (carbamate formation); at pH 7.4, physiological CO 2 tension (∼ 43 mm Hg) reduced lactate binding more markedly (∼ 75%), than lactate (50 mM) inhibited carbamate formation (∼ 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither ‘reverses’ oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb–O 2 affinity than equal increases in chloride ion concentrations. © 2006 Elsevier Inc. All rights reserved. Keywords: Haemoglobin; Lactate; Oxylabile carbamate; Carbon dioxide; Oxygen binding; Allosteric effector 1. Introduction The binding of O 2 to hemoglobin (Hb) depends on multiple interactions between Hb and erythrocytic allosteric effectors, chiefly protons and carbon dioxide (that cause the Bohr effect), anionic organic phosphates (2,3-diphosphoglycerate, DPG, in mammals) and chloride ions. These effectors bind preferentially to deoxygenated Hb and thus decrease Hb–O 2 affinity, promoting O 2 unloading in the tissues. Mammalian red cells that perfuse muscle tissues and exhibit predominantly glycolytic energy metabolism contain highly variable concentrations of lactate ions that may also bind to deoxygenated Hb (Guesnon et al., 1979) and compound the effects of proton activity (decreased pH) and CO 2 tension on O 2 delivery in the metabolising tissues. An investigation into the interactions between lactate, CO 2 and proton binding to purified Hb is called for by (a) the unexpected finding (Böning et al., 1993) that 10 mM lactate added to blood resulted in ‘negative’ oxylabile carbamate for- mation (more carbamate in oxygenated than in deoxygenated blood), (b) the marked concurrent increases in the levels of these effectors that occur under hard physical exercise in muscle venous blood, where CO 2 tensions may rise to N 70 mm Hg, lactate concentrations may exceed 20 mM and pH may fall below 7.0 (Klocke, 1987), and (c) competition between these and other effectors for binding at a small number of the 141 and 146 amino acid residues that comprise the α and β chains, respectively, of tetrameric (α 2 β 2 ) human Hb. Explicitly (see Fig. 1), polyanionic DPG binds to four β-chain residues (β1-Val, β2-His, β82-Lys and β143-His), Bohr protons react mainly at surface His residues, particularly C-terminal β146-His that accounts for about 50% of the Bohr effect (Riggs, 1988) [whereas α1-Val that until recently was considered to account for ∼ 25% of the Bohr effect may play only a minor role (Berenbrink, 2006)], and oxylabile CO 2 binding (carbamate formation) occurs at the uncharged free amino-termini of both chains (α1-Val and β1-Val). Binding of chloride, and possibly other monovalent anions like lactate (Nigen et al., 1980) is considered to occur at an α chain site (between α1-Val and α131-Ser) and at a β chain site between β82-Lys and β1-Val Comparative Biochemistry and Physiology, Part A 146 (2007) 429 – 434 www.elsevier.com/locate/cbpa ⁎ Corresponding author. Zoophysiology, Department of Biological Sciences, Univesity of Aarhus, 1131 C.F. Møllers Alle, DK 8000 Aarhus C, Denmark. Tel.: +45 8942 2599; fax: +45 8942 2586. E-mail address: roy.weber@biology.au.dk (R.E. Weber). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.12.004