ISOELECTRIC-FOCUSING OF HISTONES IN EXTREMELY ALKALINE IMMOBILIZED PHGRADIENTS - COMPARISON WITH CAPILLARY ELECTROPHORESIS

Various classes of calf thymus histones (fractions II-AS, VI-S, VII-S and VIII-S) were separated to the steady state in an extremely alkalin e immobilized pH gradient, covering non-linearly the pH 10-12 interval . Successful separations were obtained in 5%T, 4%C polyacrylamide matr ices, reswollen in 8 M urea, 1.5% Tween 20, 1.5% Nonidet P-40 and 0.5% Ampholine pH 9-11. Additionally, in order to quench the very high con ductivity of the gel region on the cathodic side, the reswelling solut ion contained a 0-10% (anode to cathode) sorbitol gradient. The best f ocusing was obtained by running the gel at 17 degrees C, instead of th e customary 10 degrees C. All major histone components had pi values b etween pH 11 and 12 and only minor components (possibly acetylated and phosphorylated forms) focused below pH 11. By summing up all bands in Arg- and Lys-rich fractions, eight to ten major components and at lea st twelve minor zones are clearly resolved. In contrast, capillary zon e electrophoresis (in a coated capillary, 7 M urea, 50 mM Tris-acetate buffer, pH 8.0) can only resolve six major fractions and two minor, b road zones.