title: Imparting mineral affinity to proteins with thiol-labile disulfide linkages.
abstract: Chemical conjugation of bisphosphonates (BPs) to proteins is an effective means to enhance binding of proteins to mineral-containing biomaterials. BPs linked to proteins with reversible (i.e., cleavable) linkages were considered desirable over the conjugates linked with stable linkages because cleavable linkages allow protein release in free form from the mineral-containing biomaterials. To explore the feasibility of creating cleavable BP-protein conjugates, an amine- and a thiol-containing BP were conjugated to the model protein Bovine Serum Albumin (BSA) with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which resulted in disulfide-linked BP-BSA conjugates. Although disulfide-linked conjugates were stable under aqueous conditions, the conjugates in solution were readily cleaved in the presence of physiological concentrations (approximately 0.3 mM) of the thiol compound, cysteine. The imparted mineral affinity as a result of BP conjugation, as assessed by hydroxyapatite (HA) binding in vitro, was lost upon cleavage of the disulfide-linked BP. The conjugates bound to HA were also cleavable with cysteine, but their cleavage rate was significantly reduced as compared to the conjugates in solution. In conclusion, disulfide-linked BP conjugates were shown to be readily cleavable by the amino acid cysteine and this resulted in the loss of imparted mineral affinity of the proteins. The proposed approach will be useful for modulating in vivo delivery of proteins implanted with mineral-containing biomaterials.
authors.affiliation: Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Geeti
authors.initials: G
authors.lastname: Bansal
authors.forename: Jennifer E I
authors.initials: JE
authors.lastname: Wright
authors.forename: Sufeng
authors.initials: S
authors.lastname: Zhang
authors.forename: Ronald F
authors.initials: RF
authors.lastname: Zernicke
authors.forename: Hasan
authors.initials: H
authors.lastname: Uludag
mesh_terms: D000818:Animals; D002417:Cattle; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004164:Diphosphonates / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D020099:Coated Materials, Biocompatible; D004164:Diphosphonates; D004220:Disulfides; D013388:Succinimides; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D003545:Cysteine