Multimolecular Forms of Thiol Proteinase Inhibitor in Human Plasma

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Abstract
The thiol proteinase inhibitor in human plasma (and serum) was separated into 3 forms [P-1(S-1), P-2(S-2), and P-3(S-3)] by linear gradient elution on a DEAE cellulose column. The individual inhibitors were purified to a functionally pure state by gel filtration on a Sephadex G-150 column, starch block electrophoresis and affinity chromatography on a trypsin-chymotrypsin-Chromagel A-2 column. The largest molecular weight inhibitor P-3 was further purified by chromatography on an anti-albumin column and by preparative polyacrylamide gel electrophoresis; the final preparation appeared homogeneous by immunoelectrophoresis and analytical polyacrylamide gel electrophoresis. Monospecific antiserum against P-3 was prepared by immunizing rabbits with purified P-3. The three forms of inhibitor showed similar pH stability curves and inhibition spectra on the proteolytic (casein substrate) and amidase (Bz-Arg-pNA, and Bz-Tyr-pNA) activities of ficin, papain, trypsin, and chymotrypsin. Moreover, the antibody against P-3 suppressed the inhibitory activities of P-1, P-2, and P-3 to similar extents in various antibody concentrations, and no spur was formed between the precipitin lines of the three forms against antiP-3 antibody by double immunodiffusion. However, the three forms differed in molecular weight and isoelectric point [mol. wt. and pI for P-1(S-1), 90,000 and 4.5 (85,000 and 4.45); for P-2(S-2), 95,000 and 4.4 (94,000 and 4.35); for P-3(S-3), 167,000 and 4.1 (150,000 and 4.0)]. P-3 was the most heat-stable, showing no decrease in inhibitory activity when heated up to 70°C for 1 h; P-1 and P-2 were stable up to 50°C. P-1(S-1) and P-2(S-2) migrated in the α1-region and P-3(S-3) in the a1-region in accordance with the difference in their isoelectric points. Thus, the term α1-TPJ is recommended for P-3(S-3), and the terms α2-TPI1 and α2 -TPI, for P-2(S-2) and P-1(S-1), respectively. These data indicate that human plasma contains three different forms of thiol proteinase inhibitor, which have the same antigenicity.