Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids

Protein adsorption characteristics of calcium hydroxyapatite (Hap) modified with pyrophosphoric acids (PPa) were examined. The PPa modified Hap particles (abbreviated as PP-Hap) possessed anchored polyphosphate (PP: P–{O–PO(OH)}n–OH) branches on their surfaces. The proteins of bovine serum albumin (BSA: isoelectric point (iep) = 4.7, molecular mass (Ms) = 67,200 Da, acidic protein), myoglobin (MGB: iep = 7.0, Ms = 17,800 Da, neutral protein), and lysozyme (LSZ: iep = 11.1, Ms = 14,600 Da, basic protein) were examined. The ζ potential (zp) of PP-Hap particles as a function of pH overlapped; zp-pH curves were independent of the concentration of pyrophosphoric acids (abbreviated as [PPa]) used for modifying Hap surface. The saturated amounts of adsorbed BSA (ΔnadsBSA) were increased three-fold by the surface modification with PPa though they were independent of the [PPa]. Furthermore, the fraction of BSA desorption was independent of the [PPa]. This enhancement of BSA adsorption onto the PP-Hap is due to the hydrogen bonding between oxygen and OH groups of the PP-branches and functional groups of BSA molecules. In the case of LSZ, a more higher adsorption enhancement was observed; the saturated amount of adsorbed LSZ (ΔnadsLSZ) for Hap modified at [PPa] = 6 mmol/dm3 was nine-fold than that for Hap unmodified. This remarkable adsorption enhancement was explained by a three-dimensional binding mechanism; LSZ molecules were trapped inside of the PP-branches. Hence, a fraction of LSZ desorption was decreased with an increase in the [PPa]; as more PP-branches are presented on the surface the higher retardation of LSZ desorption was induced. It was expected from their small size that MGB adsorb between the PP-branches as well as LSZ. However, the amounts of adsorbed MGB (ΔnadsMGB) did not vary and were independent of the [PPa] due to the small numbers of functional groups of MGB. In addition, no dependence of the fraction of MGB desorption on the [PPa] was observed. The results of zp for all the protein systems supported the mode of protein adsorption discussed. The anchored structure of the PP-branches developed on the Hap surface to provide three-dimensional protein adsorption spaces was proved by a comparative experiment that was elucidating the effect of pyrophosphate ions for BSA adsorption onto Hap.