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Biotinylated recombinant human erythropoietins: bioactivity and utility as
receptor ligand [see comments]
DM Wojchowski and L Caslake
Department of Molecular and Cell Biology, Pennsylvania State University,
University Park 16802.
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.
Volume 74,
Issue 3,
pp. 952-958,
08/15/1989
Copyright © 1989 by The American Society of Hematology
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