Abstract
To determine the effect of tether length on bone mineral affinity of fetuin-bisphosphonate conjugates. 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the lysine residues of fetuin by using five different crosslinkers that varied in length. Both the conjugation efficiency (i.e., the number of aminoBPs per protein) as well as molecular dynamics modeling of the resulting conjugates were assessed. Furthermore, the in vitro and in vivo bone mineral affinity of the conjugates were compared to one another. The tethers, whose extended lengths varied from 5.7 to approximately 136 A, were effective in conjugating aminoBP onto fetuin. Molecular dynamics modeling revealed an inverse relationship between tether length and the maximal radial density of the pendent ligand. The capacity of the conjugates to bind to various bone matrices in vitro differed significantly, as aminoBPs tethered onto fetuin via shorter cross-linkers afforded a superior affinity for various mineral matrices than those tethered via longer cross-linkers. Results from the in vivo mineral implantation studies corroborated the in vitro findings, as higher binding was achieved with the shorter conjugates. Thus, the binding capacities of the conjugates paralleled the maximal radial densities of the pendent ligands. The use of any of the chosen cross-linkers was feasible in conjugating aminoBP onto fetuin. Maximal mineral binding of the fetuin-aminoBP conjugates, however, was typically achieved using the shorter cross-linkers.
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