Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids

Abstract

Protein adsorption characteristics of calcium hydroxyapatite (Hap) modified with pyrophosphoric acids (PP a) were examined. The PP a 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 ( M s) = 67,200 Da, acidic protein), myoglobin (MGB: iep = 7.0, M s = 17,800 Da, neutral protein), and lysozyme (LSZ: iep = 11.1, M s = 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 [PP a]) used for modifying Hap surface. The saturated amounts of adsorbed BSA ( Δ n ads BSA ) were increased three-fold by the surface modification with PP a though they were independent of the [PP a]. Furthermore, the fraction of BSA desorption was independent of the [PP a]. 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 ( Δ n ads LSZ ) for Hap modified at [PP a] = 6 mmol/dm 3 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 [PP a]; 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 ( Δ n ads MGB ) did not vary and were independent of the [PP a] due to the small numbers of functional groups of MGB. In addition, no dependence of the fraction of MGB desorption on the [PP a] 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.