The adsorption of some proteins on hydroxylapatite and other absorbents used for chromatographic separations

Abstract

The absorption of bovine serum albumin and of γ-globulin (γ-G) has been studied, both with hydroxylapatite and for bovine serum albumin also with TiO 2, all in the presence of potassium phosphate buffer (pH 6.8) of a wide range of concentrations ( C Pi from 10 −3 to about 1 M). In all three cases the observed amounts of adsorbed protein can be represented over a wide range by empirical equations of the type q prot=Q C prot K 1+ C prot + exp(−K 3·C Pi) where Q, K 1 and K 3 are constants for a given system. This suggests that a common mechanism underlies the process of protein adsorption and its elution by phosphate buffer, and that this mechanism must be much more complex than previously suspected. The phosphate ions themselves are also adsorbed, both on hydroxylapatite and TiO 2, following simple Langmuir isotherms: q Pi=Q′· C Pi K 2 + C Pi The significance in physical terms of Eqn 1 is discussed in some detail. The first term represents a weak adsorption of proteins probably on negatively charged phosphate sites and independent of the phosphate buffer concentration, while the second term represents a very strong protein adsorption, invarient with protein concentration, but greatly influenced by phosphate adsorption on positively charged metal-ion sites.