Fluorescent Cytoplasm and Heinz Bodies of Hemoglobin K#{246}ln Erythrocytes: Evidence for Intracellular Heme Catabolism

H EMOGLOBIN KOLN (Hb K#{246}ln) is characterized by a point mutation in its chains, in which the f198 valine is replaced by the bulkier methionine residue.1 This substitution destabilizes the hemoglobin’s T state and increases its oxygen affinity, while at the same time loosening the heme pocket and making Hb K#{246}ln subject to heme loss and to the precipitation of granular deposits, known as Heinz bodies, on the erythrocyte membrane! Patients with unstable hemoglobin disease, such as Hb K#{246}ln, are often splenectomized in order to increase the lifetime of the circulating erythrocytes; this results in the appearance of many additional erythrocytes with Heinz bodies.3 Heinz bodies are aggregates of cytoplasmic degradation products attached to the cell’s membrane. Their mode of attachment, catabolism, and constitution are not well understood, but they appear to be the final product of a series of peroxidation reactions that begin with the oxidation of I-lbO to methemoglobin and the formation of 0, and that are the result of the structural perturbation of the heme pocket,45 Subsequent oxidation products are thought to include reversible and irreversible hemichromes, heme species in which the heme iron is covalently linked at the fifth and sixth ligand positions to the protein.6 Their presence in Heinz bodies has been established by spectrophotometry.7 Because hemichromes contain tetrapyrroles that are covalently linked to (paramagnetic) Fe3 ions, they are virtually nonfluorescent. Their absorption spectra retain the Soret band (-412 nm) ofthe heme, but the well-resolved bands of HbO, (542 and 577 nm) are replaced by poorly resolved peaks in the same wavelength region, which are superimposed on a featureless absorption rising toward the blue.61#{176}