ELBAUM zyxwv AND HERSKOVITS Dissociation of Human Hemoglobin by the Ureas and Amides. Osmotic Pressure and Light Scattering Studiest Danek Elbaum and Theodore T. Herskovits* ABSTRACT zyxwvutsrqponmlkj : The subunit dissociation of human hemoglobin by various ureas and amides has been investigated by osmo- metric and light scattering molecular weight methods. The effectiveness of these compounds as subunit dissociating agents is found to increase with increasing chain length or hydrocarbon content of the substituent alkyl groups. Nearly complete dissociation into half-molecules can be achieved before any pronounced changes are noticed in the physical properties of hemoglobin, signifying the unfolding of the in- tact zyxwvutsrqpon c~R subunits. The order of effectiveness of these two V arious neutral salts are known to dissociate hemoglobin (Benhamou et al., 1960; Rossi-Fanelli et al., 1961; Benesch et al., 1962; Guidotti, 1967; Kellett, 1971; Noren et nl., 1971 ; Thomas and Edelstein, 1972). In principle, by studying the effectiveness of various salts and reagents as subunit dissociat- ing agents and the effects of these reagents on the solubility of amino acids and peptide model compounds one should be able to predict what sort of amino acid and peptide inter- actions are important for the maintenance of the intact tetrameric structure of hemoglobin (Kirshner and Tanford, 1964; Kawahara zyxwvutsrqpo et al., 1965) or any other subunit protein. This approach has been used by various workers (reviewed by Kauzmann, 1959; Von Hippel and Schleich, 1969; Tanford, 1970) in studies concerning the factors and forces that are important for the maintenance of the folded, native structure of proteins as well as the nucleic acids (Michelson, 1963; Herskovits and Harrington, 1972). The effects of increasing hydrocarbon content of the urea, amide, and alcohol classes of denaturants on the conforma. tion of single-chain globular proteins studied in the past 5 years in our laboratory (Herskovits and Jaillet, 1969; Hersko- vits et al., 1970a-c) have now also been extended to similar studies on the single- and multi-chain hemoglobins (Elbaum et zyxwvutsrqpon a/., 1973, 1974; Herskovits et al., 1973). These studies in part also prompted the present investigation, dealing with the effects of the ureas and amides on the subunit structure of the four-chain human hemoglobin, examined by osmometric and light scattering molecular weight methods. The ac- companying paper (Elbaum et ai., 1974) examines the effects of these two classes of compounds on the denaturation of the single-chain Glycera dibranchiata hemoglobin and four-chain human hemoglobin. t From the Department of Chemistry, Fordham University, New York, New York 10458. Receioed September zyxwvutsrqp 4, 1973. This work was supported by Grant HL-14453 from the Heart and Lung Institute of the National Institutes of Health, U. S. Public Health Service. zyxwvutsr A pre- liminary report of this work was presented at the 1973 meeting of thc Federation of American Societies for Experimental Biology (Elbaum etnl., 1973). 1268 BIOCHEMISTRY, VOL. 13, NO. 6, 1974 groups of reagent as both subunit dissociating agents and as denaturing agents, at higher concentrations of reagent, is found to be the same. The observed empirical correlations found between the dissociating effects and the relative hy- drophobicities of these two series of compounds, as reflected by their binding constants, KB, suggest that the nonpolar interactions at the @-contact sites of the hemoglobin sub- units can be destabilized leading to increased subunit dis- sociation. Experimental Section Hemoglobin. Human hemoglobin was prepared from freshly drawn or spent defibrinated blood essentially according to Drabkin's (1946) procedure. The centrifuged erythrocytes were washed three times using 0.9% KC1 solutions and lysed with 10: 4 volume mixtures of distilled water and toluene in the cold followed by centrifugation to remove the cell debris and purification by passage through DEAE-Sephadex G-50 columns (Huisman and Dozi, 1965). Twice crystallized com- mercial hemoglobin (Schwarz/Mann) was purified by the same chromatographic procedure after conversion to the ferro state by passage through Sephadex G-25 columns charged with sodium dithionite (Dixon and McIntosh, 1967). All the hemoglobin solutions were stored at 4 * 2' and were usually consumed within a week after preparation. Hemoglobin Concentrution. Protein concentration was de- termined spectrophotometrically on a Cary 14 recording spectrophotometer, using the per cent extinction coefficients of 9.04 for carboxyhemoglobin at 540 nm and 8.4 for deoxy- hemoglobin at 555 nm. These values were based on the molar extinction coefficient of 4.6 X lo4 and a mol wt of 64,450 daltons, at 540 nm for the cyanmet form of hemoglobin (Drabkin and Austin, 1935), obtained by conversion with Drabkin's reagent. Reagents. All the ureas and amides were of the purest commercially available quality or spectral grade. With the exception of urea which was used without further purification (Schwarz/Mann Ultra Pure grade), all the solid ureas and amides were recrystallized from hot ethanol and were dried in a vacuum oven. 2.3-Diphosphoglycerate solutions were prepared by converting the pentacyclohexylammonium salt of the compound, purchased from Calbiochem, to the free acid form, using aqueous or buffered suspensions of Dowex 50W-X2 ion exchange resin (Benesch et al.. 1969). Osmometr, . Osmotic pressure measurements were made with a Wescon high-speed membrane osmometer utilizing Schleicher and Schuell B19 membranes. Before use the membrane compartment of the instrument was flushed with the appropriate solvent to be used and left to equilibrate overnight against the solvent at 25". The performance of the instrument and the permeability of the Schleicher and Schuell