Purification Of Binding Proteins Research Articles

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

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.

The biological and structural characterization of specific serum binding proteins for the insulin-like growth factors

The IGFs constitute an important family of skeletal growth regulatory peptides, which may play a vital autocrine and/or paracrine role in cellular growth. The role of the binding proteins in IGF physiology is quite unclear, and despite considerable progress in the past 2 years on the isolation and purification of binding proteins, many questions are still unresolved. For example, is the observed molecular heterogeneity experimentally induced, is it the result of post-translational modification of a single gene product, or are the different binding proteins products of different genes? What is the relationship between the different forms of binding proteins, and do they have different affinities for IGF-I and IGF-II? Do they have different physiological roles? It is obvious that answers to these questions are necessary in order to appreciate fully the physiological role of the binding proteins in regulating the actions of IGFs. Successful purification of different forms of IGF-binding proteins has been achieved in the last few years. This should enable the establishment of specific radioimmunoassays for each binding protein, which should allow the accurate assessment of the relative concentrations of binding proteins in various clinical or experimental states. Such studies have begun to be pursued very effectively by Baxter's group. The elucidation of the amino acid sequences of the various binding proteins will, hopefully, not only resolve the structural nature and inter-relationship of the various binding proteins, but also permit the use of recombinant DNA techniques to investigate fully, at the genetic level, the synthesis, regulation and production of the IGF-binding proteins.

Measurement of the association of cholephylic organic anions with different binding proteins

The binding of the colored cholephylic anions tetrabromosulfonphthalein (BSP), di-bromosulfonphthalein (DBSP), indocyanine green (ICG) and thymol blue (ThB) to a number of protein preparations including bovine serum albumin, human serum, rat hepatic cytosol and purified rat liver bilitranslocase has been studied by a direct spectrophotometric method. The experimentation provides extinction coefficients, dissociation constants and number of binding sites for the different complexes between dyes and the various proteins. Data obtained by this technique were in excellent agreement with those obtained on the same samples by ultrafiltration. The data presented indicate that the direct spectrophotometry applied to these dyes is simple, rapid and reproducible, making this the approach of choice during the purification of binding proteins when the binding capacity is the only useful criterion to follow the progress of the procedure.