Purification and Characterization of Insulin-like Growth Factor Binding Proteins (IGF-BP) in Amniotic Fluid

Abstract

Human amniotic fluid has been reported to contain 28-34K insulin-like growth factor (IGF) binding protein (32K-AFBP). The existence of 160K IGF-binding protein (160K-AFBP) in amniotic fluid was also demonstrated by affinity labeling studies. In this paper we describe procedures for purification of 160K -AFBP as well as 32K-AFBP from pre-term amniotic fluid and characterize these two binding proteins. IGF binding proteins were purified from batches of 500ml of pre-term amniotic fluid using ammonium sulphate precipitation, anion-exchange chromatography (Q-Sepharose) and Sephacryl S-200 HR chromatography. IGF binding activity during purification was quantitated by incubation with 125I-IGF-I and dextran-coated charcoal separation. The total recovery and purification of binding protein were 38% and 81.5-fold, respectively. The three major binding activities were eluted in fractions corresponding to a relative molecular mass of 160K, 60K and 30K when Q-Sepharose purified AFBPs were chromatographed on Sephacryl S-200 HR at neutral pH. HPLC of purified 160K binding activity on size exclusion column yielded a single peak of absorbance at 280nm, corresponding to an apparent molecular weight of 160K with three predominant peaks of binding to corresponding to molecular weights of 160K, 79K and 32K, respectively. On the other hand, purified 30K binding activity on HPLC yielded a single peak of absorbance at 280nm, corresponding to an apparent molecular weight of 32K coincident with a peak of binding to 125I-IGF-I. SDS-PAGE of HPLC purified 160K-AFBP in the presence of mercaptoethanol revealed eight bands by Coomassie Blue staining, five major bands corresponding to 160K, 78K, 69K, 54K, and 51K and three weaker bands corresponding to 45K, 36K, and 28K. The HPLC purified 32K-AFBP showed a single band of a molecular mass estimate of 32K on SDS-PAGE. These results indicate that human amniotic fluid contains at least two distinct IGF-binding proteins, and the structure of 160K-AFBP is similar to those of 150K binding protein in the circulation.