Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase hgih-performance liquid chromatography

摘要

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (a-Iactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and a-Iactalbumin (apo and holo ( + I calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (1) in the range 0-0.775 mol L- I. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Zeff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0. .For lactoferrin (pl=8-9), the electrophoretic mobility (p.) was constant and negative. highlighting a charge reversal due to phosphate adsorption. .For a-Iactalbumin (holo form) experimental IL was roughly constant and more negative than predicted. Zeff increased continuously from -4 to -II in the ionic strength range from 0.005 to 0.775 mol 1-1 , respectively. Accordingly, one to six phosphates were bound per molecule, respectively. .For lysozyme. experimental electrophoretic mobility was positive but lower than predicted. Zeff was only discrete values + 5 for I in the range 0.00 1-0.020 mol 1~(-1) and about + 3 in the range 0.050-0.500 mol 1~(-1), whereas the theoretical Z value was + 7 at pH = 7.0. Lysozyme bounds one phosphate at low ionic strength and about two -three at higher ionic strength.