J. MOE. Biol. (1986) 189, 353-359 Effects of Organic Co-solvents on the Oxygen Equilibrium of IV-Ethylmaleimide-treated Human Haemoglobin Lorenzo Cordone, Antonio Cupane and Eugenio Vitrano Istituto di Fisica dell’Universitd and G.N.S.M.X.I.S.M. Via ArchiraJi 36, 90123 Palermo, Italy (Received 26 April 1985, and in revised form 17 January 1986) We have studied the effects of monohydric alcohols and formamide on the oxygen equilibrium of native and N-ethylmaleimide-treated human haemoglobin. Comparison of t’he results obtained for the two haemoglobins gives further and compelling evidence in favour of the model, proposed recently by our group, on the role played by the solvent in the conformational equilibria of haemoglobin; moreover the results provide direct functional evidence of the relevance of the electrostatic free energy of salt bridges to the T e R equilibrium of haemoglobin. 1. Introduction This paper is one of a series aimed at investigating the role played by protein-solvent interactions in the conformational and functional properties of haemoglobin. This aim has been pursued until now by studying the reaction with oxygen, the denaturation and the dimer + tetramer equilibrium of haemoglobin in aqueous solvent perturbed by the presence of organic co-solvents (monohydric alcohols and amides) (Cordone et al., 1979a,b, 1981a,b, 1982, 1983; Cordone & Cupane, 1981; Cupane et al., 1982; Bulone et al., 1983; Vitrano et al., 1984; Cupane et al., 1986). haemoglobin. To do this we have measured the effect of monohydric alcohols and of formamide on the oxygen equilibrium of N-ethylmaleimide- treated human haemoglobin and compared the results with the analogous ones for native haemoglobin. The results for the reaction of haemoglobin with oxygen were analysed within the framework of the Monod--Wyman-Changeux model (Monod et al., 1965). Our analysis enabled us to suggest a model according to which the solvent affects the tense = relaxed equilibrium of haemoglobin through contributions linearly related to the inverse bulk dielectric constant of the medium (bulk electro- static contributions) and hydrophobic contributions arising from the exposure to the aqueous solvent of hydrophobic surfaces, following the T --, Rt transition (non-bulk electrostatic contributions). As is well known, NES haemoglobin is a chemically modified haemoglobin in which the sulphydryl group of Cys93/Y? has been reacted with N-ethylmaleimide (Riggs, 1961; Benesch & Benesch, 1961); this brings about the disruption of the normal salt bridge between His146fi and Asp94P in the deoxy T conformation (Perutz et al., 1969). We observe that bulk electrostatic contributions are 20% lower for NES haemoglobin than for human haemoglobin, whereas non-bulk electrostatic (hydrophobic) contributions are the same for both haemoglobins. 2. Materials and Methods Here we have studied the dependence of these two contributions upon the number of salt bridges (Perutz, 1970) that stabilize the T conformation of Human haemoglobin from the blood of healthy donors was prepared as described (Cordone et al., 1979a). Haemoglobin preparation was started immediately after blood was drawn. Concentrated oxyhaemoglobin stocks were kept frozen in liquid nitrogen. Freezing was performed by dripping the haemoglobin solution into a container filled with liquid nitrogen (Di Iorio, 1981). Samples of the stock were melted as needed. Control measurements for oxygen uptake were performed with each portion; the results were extremely reproducible. t Abbreviations used:T (tense),R (relaxed) statesof NES haemoglobin was prepared by adding N-ethyl- haemoglobin; MWC, Monod-Wyman-Changeux; NES maleimide (Pierce) to the melted stock in the ratio haemoglobin, N-ethylmaleimide-treated human (haemoglobin tetramer)/iV-ethylmaleimide = 1 : 5 (Riggs, haemoglobin. 1961); the NES haemoglobin stock was kept for 36 h at (*22-2836/86/1(~353-7 $03.00/O 353 0 1986 Academic Press Inc. (London) Ltd.