PARTIAL PURIFICATION OF LOW MOLECULAR WEIGHT NON-SUPPRESSIBLE INSULIN-LIKE ACTIVITY FROM HUMAN PLASMA: DEMONSTRATION OF THE PRESENCE OF MULTIPLE FORMS

Abstract

An alternative procedure for the separation of insulin-like growth factors from plasma, avoiding the harsh conditions of acid–ethanol extraction, suggested that several factors which have activity in the isolated rat adipocyte assay may be present. The initial step consisted of ion-exchange chromatography of an enriched Cohn fraction (IV-1) on SP-Sephadex using ammonium acetate buffers. The major activity appeared in a fraction eluted at pH 7·1–9·4. When this fraction was subjected to gel filtration on Sephadex G-75 in 1% formic acid, most of the activity was recovered in a fraction of molecular weight of 5000–8000, but up to 25% of the activity applied remained in a much higher molecular weight form. The active material of low molecular weight was recovered at a considerably greater yield compared with that obtained by the more conventional acid–ethanol extraction followed by gel filtration on Sephadex G-75. The fraction was further purified by DEAE–Sephadex chromatography and isoelectric focussing. Non-suppressible insulin-like activity (NSILA) of pI 4·8 and 6·0 were the major species detected. The results were in contrast with those obtained using acid–ethanol extraction of the Cohn fraction, where the major species were of pI 6·0 and 8·0. The presence of multiple forms of NSILA would thus appear to justify the use of alternative methods of purification.