Electr’on Spin Resonance Studies of the Denaturation of Oxy- and Methemoglobin

The electron spin resonance technique shows that the heat denaturation of oxyhemoglobin involves (a) oxidation to methemoglobin or related material (high spin ferric), (b) conversion to low spin ferric species in an irreversible step, and (c) further changes in these low spin species under drastic conditions. The last two occurrences are observed with methemoglobin. The electron spin resonance spectra obtained upon heat denaturation are ascribed to at least two low spin ferric complexes between hemin and denatured globin. The technique is used to follow the irreversible heat denaturation of oxyand methemoglobin and to obtain transition temperatures for denaturation. Transition temperatures in the presence of organic solvents, solutes, and salts and in the presence of a few inorganic salts are obtained, and from these molar changes in transition temperatures are estimated. In some cases, the transition temperature is lowered to 23’ and below. Organic compounds, except for glycerol and sucrose, all lower transition temperatures, and their effectiveness as denaturants increases as their hydrophobic character increases, with the notable exception of guanidinium chlorides. Among inorganic salts, anions which salt in proteins, such as iodide and thiocyanate, decrease heat stability whereas those which salt out proteins, such as sulfate and citrate, increase stability over that found in water. These data are discussed with regard to protein-solvent interactions. A few organic solutes, notably methanol, formamide, phenol, and urea, can bring about a change in methemoglobin, characterized by a particular electron spin resonance spectrum. The species responsible for the spectrum appears to represent an internal complex between hemin and a slightly perturbed or denatured form of globin.