Isoelectric point of free and adsorbed cytochrome c determined by various methods.

The purpose is to determine the isoelectric point (IEP) pI of cytochrome c (cytC, a globular haemoproteid) adsorbed on montmorillonite (MM, plate-like colloid particles) by microelectrophoresis and to compare the pI value with pI9.44 measured by isoelectric focusing in gel with covalently linked ampholytes, and with pI10.0-10.6 of free cytC globule calculated by three programs for pH-dependent net charge nz using the crystallographic structure of cytC. The pH-dependence of the electrophoretic mobility μ(pH) in the range pH 6-11 shows out that IEP of cytC-MM particles appears at pH 9.35. The near courses of μ(pH) and nz(pH) reveal that the pH-independent negative charge of the MM substrate is hydrodynamically shielded by the adsorbed protein globules. The nearness of IEP and pI allows attributing IEP value of cytC-MM particles to the isoelectric point pI of cytC. A short survey for pI of cytC reported in the literature since 1941 shows out that pI is dispersed in the range pH 9.0-10.65 although cytC is used now as pI marker with well known IEP; the reason for that and the imperfections of the employed methods are discussed.