Purification and Characterization of a Low Molecular Mass Cysteine Proteinase Inhibitor from Human Amniotic Fluid

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Abstract
The human low molecular mass cysteine proteinase inhibitor was purified in good yield from amniotic fluid, using ultrafiltration through 100-kDa [kilodalton] and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54 and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsin B, C and H, the cathepsin B-like enzyme from B16 melanoma cells and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2+-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI [isoelectric point] values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5 and 5.2, all of which inhibited papain.

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