Identification and High Yield Purification of Insulin-Like Growth Factors (Nonsuppressible Insulin-Like Activities and Somatomedins) from Human Plasma by Use of Endogenous Binding Proteins*

Abstract

The insulin-like growth factors (IGF) of human serum, somatomedins A and C, and nonsuppressible insulin-like activities (NSILA) I and II are available in only a few laboratories and in very limited quantity. In the present study we have identified three peptides with insulin-like activity and have partially purified two of them from human plasma by a high yield method that utilizes the natural affinity of these peptides for their binding protein(s). Human plasma was ultrafiltered at neutral pH and the insulin-like growth factors, which were associated with high molecular weight binding protein(s), were retained, while insulin and other low molecular weight polypeptides were eliminated. The retentate was acidified to dissociate the peptides from their binding protein(s) and the ultrafiltration was repeated. The dissociated peptides were collected in the ultrafiltrate. Further purification was obtained on gel filtration on Sephadex G-75. Two peaks of activity were observed with apparent molecular weights between 6,000–10,000. The more retarded peak, comprising 80% of the activity, was further resolved by isoelectric focusing into two sharp bands. The major fraction (75%) had an isoelectric point of 6.5, similar to somatomedin A; the minor fraction (25%) had an isoelectric point of 8.3, similar to somatomedin C and the recently purified NSILA I and II (also known as IGFs I and II). Both fractions were active in the insulin and IGF radioreceptor assays. The specific activities of these fractions were 28 and 25 mU/ml insulin-like activity, respectively. Both fractions had insulin-like and somatomedin bioactivities, but only the pi 8.3 fraction contained immunoreactive somatomedin C. The overall purification was 20,000-fold from plasma and the recovery of activity was nearly complete. This purification scheme will allow purification of substantial quantities of the insulin-like growth factors.