Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid.

Abstract

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl-Sepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%. Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.