Liberation of Immunoreactive Somatomedin-C from Its Binding Proteins by Proteolytic Enzymes and Heparin*

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Abstract
Most of the somatomedin in serum exists as a high molecular weight complex (≈140,000) composed of binding protein subunits and small molecular weight somatomedin. These binding proteins may interfere with measurements of the somatomedins by various radioligand assays. In a companion paper, we reported that when serum is incubated at neutral pH the detectability of the somatomedin-C (Sm-C) is increased. Using gel chromatography, however, it was not possible to demonstrate release of free Sm-C from the macromolecular complex. In the studies reported here, incubation of heparinized plasma at pH 7.4 followed by gel chromatography in a heparincontaining buffer caused 70-80% of the immunoreactive Sm-C to shift from the γ-globulin region to a molecular weight which approximates that of free Sm-C. This conversion is a timedependent process which is inhibited by the proteolytic enzyme inhibitor antipain. Similar changes in the elution profile of Sm-C were observed when heparinized plasma was acidified and chromatographed in heparin. These findings suggest that 1) at neutral pH, serum proteolytic enzymes reduce the affinity between small molecular weight Sm-C and its binding proteins. 2) At acid pH, a similar effect is observed. 3) In the presence of heparin, reassociation of these components does not occur. These results suggest a possible mechanism whereby the somatomedin macromolecular complex could be disrupted so that small molecular weight somatomedin is made available to tissues.