The Effect of Different Isolation Procedures on the Yields of Insulin-Like Growth Factors from Human Plasma

Abstract

Various procedures for the isolation of insulin-like growth factors (IGF's) from human plasma were evaluated with emphasis upon the yields of each species obtained. The procedures which were evaluated as first steps from plasma and mixed Cohn fractions included acid-ethanol extraction, heat coagulation, ion-exchange chromatography and gel filtration. Certain two-step combinations of these techniques were also evaluated in order to obtain higher fold-purification. Fractions obtained were tested initially using a competitive binding protein assay and then by radioimmunoassay (RIA) for IGF I and II. The results indicated that acceptable yields of IGF I and II were obtained by initial gel filtration of acidified plasma on Sephadex G-75 at pH 2.5 followed by chromatography on S.P. Sephadex C-25. Isoelectric focusing of the active fraction thereby obtained under these conditions and subsequent R.I.A. indicated total recoveries of IGF I and II of about 23 and 96 mU/litre respectively. Peak recoveries of IGF I were obtained in a fraction of pI 7.6-8.2 and IGF II between pI 5.9 and 6.9. The order in which the two steps of gel filtration and ion exchange chromatography were carried out appeared to alter the distribution of IGF's obtained. When ion exchange chromatography was used as the first step, most of the activity was obtained in fractions of pI 4.4-5.9 and was not immunoreactive IGF I or II.

Isolation and partial characterization of IGF-like peptides from Cohn fraction IV of human plasma

“…mass units lower than the calculated value, an as yet unexplained difference. It should be noted that the pi obtained by us for both peptides (IGF-I: 8.7, IGF-II: 6.7) is somewhat higher than has mostly been reported (IGF-I be¬ tween 8.0 and 8.7, IGF-II: 6.2-6.5) (9,11,39,42).…”

“…mary structure has furthered the insight into their structural relationship with other insulin-like sub¬ stances and has been the basis for the isolation of cDNAs encoding them (38). The presence in plasma and in Cohn fraction IV of other fractions with IGF-like activity, as mea¬ sured by bioassay or RRA, has been recognized (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)39). The procedures used for full purification may have induced selective enrichment of certain peptides and loss of others.…”

“…After extraction, the preparations were separated by isoe¬ lectric focusing (IEF) on a preparative flat-bed (LKB): preswollen Ultradex (LKB) was mixed with at a maxi¬ mum 500 mg proteins and ampholines (respectively, 1.8 ml, pH 5-7; 1.8 ml, pH 7-9, and 4.0 ml, pH [9][10][11] and evaporated to a suitable water content and a final volume of 100 ml. Focusing was done overnight, initially with 17.5 mA and a limitation of the power to 7.5 W. The fractions were eluted with water and the pH was measured at 4°C.…”

“…The heterogeneity of insulin-like growth factors (IGF, somatomedins) in extracts of human plasma or serum has been noted since the earliest attempts to isolate and characterize them (1)(2)(3). While side fractions with somatomedin and/or insulin-like ac¬ tivity were observed by many authors (4)(5)(6)(7)(8)(9)(10)(11), the focus has been on the two main acid extractable-somatomedins: IGF-I (12,13) and IGF-II (14).…”