Identification and purification of a novel serine proteinase inhibitor.

We report the identification, purification, and partial amino acid sequence of a novel serine proteinase inhibitor which is present in extracts from human placentas and in the cytosolic fraction of the leukemic cell line K562. Extracts from these tissues exhibited time-dependent inhibition of the serine proteinase thrombin. This activity was not accelerated by heparin and corresponded to a protein which formed a 67-kDa complex with 125I-thrombin. The complex was stable on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cleaved and functionally inactive form of the protein was purified from placental tissue by chromatography on DEAE-Sepharose, followed by affinity chromatography on thrombin-Sepharose. Antibodies raised against the placental protein recognized the inhibitor from K562 cells and placental extract. Western blotting experiments using the antibody showed that the uncleaved inhibitor has a molecular mass of 38 kDa. Amino acid sequencing was performed on the purified protein. Sequences of peptides resulting from digestion with cyanogen bromide followed by Endoproteinase Lys-c confirmed that this is a novel inhibitor with significant homology to the serpin family.