Solution studies on heme proteins: Circular dichroism and optical rotation of Lumbricus terrestris and Glycera dibranchiata hemoglobin

Abstract The circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of the multi-chain hemoglobin of the earthworm, Lumbricus terrestris, are presented and compared to the spectra of the single chain and four-chain Glycera dibranchiata and human A hemoglobins in both the ferro and ferri heme oxidation states. In Fe2+ oxy form L. terrestris hemoglobin exhibits positive dichroic bands at 258,412, and 560 nm and negative bands at 288,330, 520, and 578 nm. Except for the presence of a positive CD band at 577 nm, corresponding to the 578 nm band of L. terrestris oxyhemoglobin, human oxyhemoglobin has similar CD spectra in the same near-ultraviolet to visible wavelength region. On the other hand, all the resolved dichroic bands or extrema of G. dibranchiata hemoglobin at 280, 295, 345 and 540 nm are negative, save for the last band at 575 nm, which is positive. These differences seem to result from the changes in the heme environment and the geometry, and coordination state of the central heme iron in these proteins. While the changes in the oxidation state to the Fe3+ form of these hemoglobins seem to produce significant changes in the position and amplitude of most of the observed CD bands, including a change in the sign of L. terrestris cyanomethemoglobin at 525 and 575 nm, the largely negative character of the CD spectra of the G. dibranchiata cyanomethemoglobin (with negative CD bands shifted to 270, 297, 364, 427, and 548 nm) seems to be preserved. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region are found to be largely unaltered on changes in oxidation states and ligand binding. The CD spectrum of L. terrestris oxyhemoglobin is thus characterized by CD bands centering at 218, 208 and 193 nm, with mean residue ellipticities [Θ]λ = −15 750, −14 900, and 33 100 deg. · cm2 · dmole−1, and the ORD spectrum with extrema at 233 and about 200 nm with mean residue rotations, [m′]λ of −5430 and 33 000 deg. · cm2 · dmole−1. The corresponding CD and ORD amplitudes of G. dibranchiata oxyhemoglobin are found to be, respectively, [Θ]λ = −24 350, −23 100, and 61 600, and [m′]λ = −9180, and 48 400 deg. · cm2 · dmole−1. Estimates of apparent helix content obtained with the recently published ORD and CD reference parameters by Chen et al., based on X-ray structural and solution data of various proteins, indicate rather low helical folding of 33 to 45% for L. terrestris hemoglobin and 70 to 75% helix for G. dibranchiata hemoglobin. The latter estimate is similar to that obtained on human hemoglobin A. The possibility that the low estimates of helix content of L. terrestris hemoglobin are caused by spectral distortions and absorption flattening due to Mie scattering of this high molecular hemoprotein was considered, and ruled out, largely on the basis of light scattering and concommitant ORD and CD measurements as a function of pH. Light scattering measurements at 630 nm confirmed the mol. wt of approx. 3.1 · 106 at pH 7.0. Dissociation of the relatively large subunit organization of this protein in the alkaline region, at pH 10.1 to 10.4, to about a tenth to a twelfth of the original molecular weight was found to have relatively little or no effect on ORD and CD spectra. No increases in the amplitudes and significant shifts in the wavelengths of the 233 and 200 nm ORD and 218, 208, and 193 nm CD band were found, suggesting that spectral distortions are of minor importance regarding the ORD and CD spectra, and estimates of helix content of L. terrestris hemoglobin.