Proteinase inhibitory assays of serum using high-performance size exclusion chromatography

A size exclusion column (Spherogel TSK-2000 SW) was utilized in a high-performance size exclusion chromatographic assay to determine the proteinase inhibitory capacity of human sera. Values from assays using this technique agreed well with the standard spectrophotometric inhibitory assays. Nanogram to milligram amounts of protein, namely, alpha 1-proteinase inhibitor, elastase, trypsin, chymotrypsin and their corresponding complexes with the inhibitor, were fractionated in less than 15 min. The nitrated or oxidized alpha 1-proteinase inhibitor was shown to retain its ability to form stable complexes with trypsin or chymotrypsin; however, they lost the inhibitory activity against elastase and instead they behaved as common protein substrates for this enzyme. The present chromatographic procedure was unable to detect any peptide released when the native inhibitor and any of the proteinases reacted to form a complex. Moreover, dissociation of the alpha 1-proteinase inhibitor--elastase complex in an alkaline pH did not result in the formation or release of any peptide.