title: Biotinylated recombinant human erythropoietins: bioactivity and utility as receptor ligand.
abstract: Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.
authors.affiliation: Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
authors.forename: D M
authors.initials: DM
authors.lastname: Wojchowski
authors.forename: L
authors.initials: L
authors.lastname: Caslake
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D051379:Mice; D011869:Radioligand Assay / Q000379:methods; D011956:Receptors, Cell Surface / Q000032:analysis*; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000378:metabolism; D019809:Streptavidin; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D001710:Biotin; D019809:Streptavidin