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

• Published in: Colloids and surfaces, B, Biointerfaces B, Biointerfaces, 2019-02, Vol.174, p.87-94
• Main Authors: Hristova, Svetlana H., Zhivkov, Alexandar M.
• Format: Article
• Language: English
• Subjects: Cytochrome c; Isoelectric focusing; Microelectrophoresis; Montmorillonite; Protein adsorption; Protein electrostatics
• ISSN: 0927-7765; 1873-4367
• DOI: 10.1016/j.colsurfb.2018.10.080

[Display omitted] •Adsorption of cytochrome c (cytC) on montmorillonite (MM) colloid particles.•Employed methods: micro-electrophoresis, isoelectric focusing, protein electrostatics.•Isoelectric point of cytC-MM particles coincide with that of free cytC.•There are no reliable techniques for isoelectric point of free proteins.•The isoelectric point of cytochrome c in literature has inaccuracy of 1.7 pH-units. The purpose is to determine the isoelectric point (IEP) pIμ of cytochrome c (cytC, a globular haemoproteid) adsorbed on montmorillonite (MM, plate-like colloid particles) by microelectrophoresis and to compare the pIμ value with pIf9.44 measured by isoelectric focusing in gel with covalently linked ampholytes, and with pInz10.0–10.6 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 pIf allows attributing IEP value of cytC-MM particles to the isoelectric point pIμ 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 pIf marker with well known IEP; the reason for that and the imperfections of the employed methods are discussed.