Placental transferrin receptor. Evaluation of the presence of endogenous ligand on specific binding.

The effect of endogenous ligand on transferrin binding to receptor was investigated using a Triton X-100 extract of acetone powder from human placentae as the source of transferrin receptor. This extract contained 2.0 +/- 0.4 micrograms of endogenous transferrin per mg of protein. The studies showed that it is necessary to account for the concentration of endogenous transferrin when calculating the specific binding and the association constant (Ka) of transferrin to the receptor. The attempts to remove endogenous transferrin from the extract by washing with agents such as 2.0 M KSCN and glycine/NaOH, pH 10.0, with 1 M NaCl or by immunoabsorption with anti-transferrin were unsuccessful in that substantial amounts (10%) of endogenous ligand remained. Under the assumption that endogenous and exogenous transferrin have similar affinity of binding to the receptor, the effect of endogenous ligand on specific binding could be accurately determined by measuring the amounts of transferrin in the placental extract by radioimmunoassay and then accounting for it in the total transferrin concentration in the experiments. The Ka value corrected for endogenous transferrin present, 2.39 +/- 0.35 X 10(9) M-1 (n = 6), was approximately 3 times higher than the value obtained without consideration of endogenous transferrin, 0.87 +/- 0.20 X 10(9) M-1 (n = 5). From these studies, it appears that a true Ka value for ligand-receptor binding cannot readily be determined experimentally in the presence of substantial amounts of endogenous ligand but that the endogenous ligand must be quantitatively measured and the amounts present corrected for in the calculation of the Ka value.