The effect of different isolation procedures on the yields of insulin-like growth factors from human plasma

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.