of these amino acids carry side-chain carboxyl or amino Protein adsorption isotherms are often described by the Langgroups, and these may remain free and exposed to the solvent muir equation. Protein adsorption is generally a cooperative prowhen the protein is in solution. Therefore, they can dissociate cess. Although the simple Langmuir equation does not normally in aqueous solution at a suitable pH, resulting in COO 0 and include cooperativity, the Hill plot can represent cooperativity, NH / 3 ions covalently attached to the protein macromolecule.
including the degree of cooperativity. Our studies of the adsorption
The degree of ionization of these groups is not greatly influof lysozyme, albumin, transferrin, and lactoferrin onto hydroxyapenced by their incorporation into the large molecule (62).
atite suggest that cooperativity can be described as positive (n ΓΊ
The carboxyl group tends to ionize at pH values over about 1.5), partially positive (1 Γ΅ n Γ΅ 1.5), partially negative (1 Γ΅ n 4 and the amino group at below about 12 (46). Thus, in Γ΅ 0.5), and negative (n Γ΅ 0.5). There are three stages (i, ii, and iii) as protein concentration increases, similar to the cooperativity acid solution, a typical protein becomes positively charged observed in the interaction between oxygen and hemoglobin. The because of the presence of NH / 3 and COOH groups, and in number of stages is determined by the structural rigidity of proteins basic solution it is charged negatively because of NH 2 and and by the lateral interactions between proteins and between pro-COO 0 groups. When pH equals pI, the net charge is zero. tein and adsorbent. Our Hill plots show that stage ii has the lowest This corresponds to the presence of equal numbers of oppodegree of cooperativity, stage iii has the highest, and stage i is in sitely charged groups on the protein. Such a neutral structure between, for most cases. In stage i the low degree of cooperativity of charged groups is called a dipolar ion, or a zwitterion is highly consistent with the linear range in the adsorption isotherm (47). At pH's near the p I, both NH / 3 and COO 0 groups when protein solution concentration is low. These results provide are present, so that the net charge is small (Fig. 1).
useful information for the design and optimization of analytical adsorption chromatography. The factors determining the degree
Calcium hydroxyapatite (HA) , Ca 5 OH (PO 4 ) 3 , is the of cooperativity include protein conformation, pI, and solution mineral prototype for both bones and teeth, and it is often pH. We now quantitatively understand the cooperative adsorption considered a suitable material for making medical implants. of these proteins on hydroxyapatite, the degree of cooperativity, HA has been used as a support for the chromatographic and how to control the adsorption process. α§ 1998 Academic Press separation of biological macromolecules ( 4-9). Of interest in biotechnology, it is used as a carrier for genes and for enzyme immobilization ( 10). There has been substantial multiple interactions. In enzymes, the term allostery often 104