1. Erythrocyte hemins, extracted with acid-acetone, exhibited multiple components on two chromatographic systems. However, this heterogeneity can be explained by various physicochemical properties of hemin in solution and does not imply that heme, when attached to globin in the native hemoglobin molecule, is heterogeneous.
2. The presence of a non-polar fraction may be accounted for by esterification of protohemin as a result of reaction with small amounts of methanol contaminating the acetone and/or the presence of some un-ionized protohemin molecule in solutions of low pH.
3. The presence of poorly soluble fractions is probably the result of polymerization of the hydroxyhemin molecule into so-called β- and γ-hematins.
4. The formation of mesohemin is considered unlikely, but the formation of deuterohemin or hematohemin cannot be excluded.
5. No changes in hemin chromatographic patterns were noted in disease.
6. It is necessary to evaluate reports concerning chromatographic hemin heterogeneity in the light of the artifacts described above before attributing physiologic or pathogenetic significance to the findings.