Antibacterial activity and hydroxyapatite adsorption ability of Ser(p)-Histatin 5

Abstract

Objectives: Histatin 5 (H5) is a natural antimicrobial peptide from human salivary glands. The objective of this study is to compare the antibacterial activity and the hydroxyapatite adsorption ability of H5 and SP-H5 (adding one phosphorylated serine). Methods: Two-fold serial dilutions of H5 and SP-H5 were prepared in 96-well cell culture plates, with BHI (brain-heart-infusion) as control. 10μl streptococcus mutans (S. mutans) suspensions were added up to 106 CFU/ml. After incubation anaerobically 48 h, minimal inhibitory concentration (MIC) was tested with microplate reader. Minimal bactericidal concentration (MBC) of peptides was determined after suspension cultured on blood agar. The adsorption quantity of H5 and SP-H5 with hydroxyapatite particles was determined by Micro BCA method. 5nmol/ml of H5 and SP-H5 (N=12) were prepared, wherein one half (n=6) were mixed with hydroxyapatite particles and the other half (n=6) were not. Working reagent was added into 96-well plate, then bovine serum albumin standard solutions and peptide supernatants were added, respectively. The absorbance (A) was measured by microplate reader to determine linear function C = (A-a)/b (C, concentration of peptide solution; a and b, constant). Final concentration of peptide solution mixed with hydroxyapatite (Cf) and initial concentration of peptide solution (C0) were calculated using absorbance of peptide solution mixed with hydroxyapatite (Af) and absorbance of initial peptide solution (A0). T-test was used to compare the outcome of Cf-C0 in H5 and SP-H5 groups. Results: MIC and MBC of H5 and SP-H5 were both 2μmol/ml and 4μmol/ml, respectively. SP-H5 shew significantly higher adsorption property with hydroxyapatite (p<0.05). Conclusions: SP-H5 has similar antibacterial activity to inhibit S. mutans as H5; while SP-H5 has stronger hydroxyapatite adsorptive ability in order to achieve better retention on enamel surface.