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Abstract Anomalous boundaries resulting from the binding of a buffer constituent give the appearance of electrophoretic inhomogeneity to pure crystalline native monomeric cytochromes c from 10 species (man, horse, hog, guanaco, Pekin duck, pigeon, turkey, tuna, a moth (Samia cynthia), and the screw worm fly). The electrophoretic patterns depend on the particular cytochrome c used and its concentration, as well as the nature of the anion and its concentration. At pH 6.9 the order of increasing cathodic mobility of the protein is phosphate l chloride ≤ iodide l sulfate l cacodylate. Cations have no effect. The isoionic points of all the cytochromes c tested are at pH 10.04 ± 0.04 and are identical with the isoelectric points in the nonbinding tris(hydroxymethyl)aminomethane-cacodylate buffer. At pH values removed from the isoelectric region the electrophoretic mobilities of various cytochromes c differ, as expected from the different net charges calculated from the amino acid compositions of the proteins. The changes in the chromatographic mobility of cytochrome c on cation exchange resins at different ionic strengths are probably caused by the same buffer reaction phenomena which affect the electrophoretic behavior of the protein. The remarkable evolutionary constancy of isoionic points and net charges at neutral pH is related to the importance of those physicochemical parameters in all cytochrome c physiological reactions and may represent a case of compensating evolutionary variations.
Barlow et al. (Fri,) studied this question.