On the Heterogeneity of Beef Heart Cytochrome c. IV. Isoelectric Fractionation by Electrolysis in a Natural pH-Gradient.

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Abstract
The occurrence of multiple molecular forms (Cy I - Cy IV) [cytochrome-c subfractions] of monomeric beef heart cytochrome c previously shown 1,2 was confirmed by the method of stationary electrolysis for isoelectric focusing of proteins. Their isoelectric points (pI) were determined at 4[degree]C. In addition, subfraction Cy II (as defined by disc and moving boundary electrophoresis) was resolved into 2 components (Cy II1 and Cy II2) which differ only by 0.03-0.04 pH units in their pI. The resolution of Cy II1 and Cy II2 agrees well with the resolution of Cy II previously obtained by chromatography on cation-exchange resin (Duolite CS-101). Thus, additional evidence is given in support of the view that Cy II statistically should be composed of isomers that possess the same number of amide groups, but with 1 of them positioned on different carboxyl groups. The limitations of the method of stationary electrolysis for isoelectric focusing and pi measurements of very basic proteins are discussed.