Site-specific rate constants for iron removal from diferric transferrin by nitrilotris(methylenephosphonic acid) and pyrophosphate

Abstract

The kinetics of iron removal from human serum diferric transferrin by nitrilotris(methylenephosphonic acid) (NTP) and pyrophosphate (PPi) have been studied in 0.1 M, pH 7.4, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonate buffer at 25 degrees C using urea/polyacrylamide gel electrophoresis. The four microscopic rate constants required for a complete description of iron removal from the two transferrin metal-binding sites have been measured at 100 mM concentrations of NTP and PPi. There is very good agreement between the rate constants determined by electrophoresis in this study and the corresponding rate constants determined spectrophotometrically for monoferric transferrins that have been labeled at the empty binding site with substitutionally inert Co(III). The results validate the use of cobalt-labeled transferrins as models for kinetic studies on iron removal from diferric transferrin.