A preparative scheme has been developed for the purification of a trace protein in human serum exhibiting nonsuppressible insulin-like activity (NSILA). This scheme consisted of (a) adsorption chromatography of serum utilizing the sulfonic acid polystyrene resin, Dowex 50, at pH 6.8; (b) (200 gel filtration at pH 8.9; and (c) acrylamide gel electrophoresis in a discontinuous preparative system. Throughout all procedures, NSILA fractionated as a single molecular species approximating 90,000 mol wt. The purified protein exhibited a single band by disk gel electrophoresis, an isoelectric pH approximating 6.2, doublet bands of 90,000 mol mt by analytical sodium dodecyl sulfate gel electrophoresis, and a biologic specific activity approximating 50 mU/mg. Serum somatomedin (sulfation factor) activtiy did not fractionate with NSILA in this scheme, and partially purified NSILA did not stimulate radiosulfate uptake into hypophysectomized rat costal cartilage. This protein appears to represent the major constituent of serum NSILA: its purification and partial characterization provides the first step towards elucidation of its metabolic role.
P L Poffenbarger