E. A. Rachmilewitz

A new natural anti-alpha-galactosyl IgG antibody (anti-Gal) was found to be present in high titer in the serum of every normal individual studied. The antibody was isolated by affinity chromatography on a melibiose-Sepharose column. The reactivity of the antibody was assessed by its interaction with alpha-galactosyl residues on rabbit erythrocytes (RabRBC). The specificity was determined by inhibition experiments with various carbohydrates. The anti-Gal interacts with alpha-galactosyl residues, possibly on glycolipids of human RBC (HuRBC), after removal of membrane proteins by treatment with pronase. In addition, the anti-Gal bind specifically to normal and pathologically senescent HuRBC, suggesting a physiological role for this natural antibody in the aging of RBC. The ubiquitous presence of anti-Gal in high titers throughout life implies a constant antigenic stimulation. In addition to the theoretical interest in the antibody, the study of the anti-Gal reactivity seems to bear immunodiagnostic significance. Decrease in the antibody titer was found to reflect humoral immunodeficiency disorders.

Abstract All heme proteins containing mononuclear high spin ferric heme, when examined at low temperatures (near 1°K), exhibit X-band electron paramagnetic resonance (EPR) absorptions extending from near g = 6 to g = 2 which arise from transitions of the lowest Kramers doublet. Meaningful absolute quantitation of high spin ferric EPR spectra cannot be made other than from experiments at very low temperature or from a knowledge of the zero field splitting. The characteristics of the EPR spectrum may be used to describe the symmetry of the heme. The incorporation of hemin into a protein constrains the heme in such a manner that there is a departure from tetragonal symmetry toward rhombic (gx ≠ gy). In these cases, the resonance absorption derivative near g = 6 is either broadened or split into two resolvable g values dependent upon the interaction of the protein with the heme. The greater the constraint on the heme imposed by the protein, the greater will be the departure from tetragonality. Thus, the EPR of high spin heme proteins can be used as a protein conformational probe.

Abstract Oxyhemoglobin either spontaneously or under the influence of external oxidants is converted to the high spin ferric protein (acid methemoglobin) which can be studied both optically and with electron paramagnetic resonance (EPR). The rate of ferricyanide oxidation is comparable for oxyhemoglobin A, oxyhemoglobin H (β4), and oxy α chains. p-Mercuribenzoate (PMB) derivatives of oxy α and oxy β chains react faster with this reagent than do the nonderivatized proteins. With time, the high spin form of each hemoglobin preparation is converted to various low spin forms, heretofore collectively called Hemichromes are low spin derivatives of ferrihemoglobin, brought about through discrete reversible and irreversible changes of protein conformation so that atoms endogenous to the protein are now bound as the sixth ligand of the heme iron. The rate of formation of the first hemichromes from the obligatory high spin ferric protein is very slow with ferrihemoglobin A, faster with ferrihemoglobin H, and faster still with ferric α chains. This first hemichrome can be reversibly converted to deoxyhemoglobin by reduction to the hemochrome followed by anaerobic dialysis, or it can be converted quickly to oxyhemoglobin by reacting the hemochrome with CO which is then photolyzed in the presence of O2. It has been demonstrated that ferrihemoglobin cyanide prepared from high spin ferrihemoglobin H differs in structure from ferric hemoglobin cyanide prepared from the hemichrome of hemoglobin H, although the optical and EPR spectra of the two compounds are identical. With time, or under the influence of protein denaturants such as urea or salicylate, the hemichrome that can be reconstituted to oxyhemoglobin is converted to different hemichromes which are nonrenaturable to the oxy protein. Hemin added to whole globin yields high spin ferrihemoglobin A, whereas hemin added to globin prepared either from α or β subunits yields hemichrome.