Serpines A3 bovines

Abstract

The SERPINA3 or α1-antichymotrypsin is a glycoprotein of the serpins superfamily, a functional inhibitors group of serine and cysteine peptidase-like. In vivo, human SERPINA3 is a plasma protein inhibiting cathepsin G, chymotrypsin and chymase. The aim of my study, is the purification and characterization of SERPINA3 presumed to be a specific inhibitor of caspase 3. The purification of the inhibitor is followed by a structure-function study to understand its biological role. To do this, a purification protocol was developed. The SERPINA3 was thus purified from human serum by ammonium sulfate fractionation between 50 and 80 % saturation, followed by a series of chromatography, the first is an affinity chromatography on a HiTrap Blue Sepharose 5 ml (1.6 x 2.5 cm) and the second is an anion exchange chromatography on a Q-Sepharose Fast Flow (2.5 x 10 cm), and finally the fractioned pool with a high antichymotrypsin inhibition is re-injected a second time on the same chromatographic supports with the same order as upper. Then, the identification of the molecular weight by SDS-PAGE of the inhibitor purified, the identity with an antibody directed against bovSERPINA3 by Western blot, identification of the protein by mass spectrometry by MALDI-TOF and LC-MS/MS, the protein polymorphism by isoelectric focusing (IEF) on a two-dimensional gel electrophoresis (2DE) and enzyme activity against bovine pancreatic trypsin, bovine chymotrypsin, human neutrophil elastase and human caspase 3 were determined. Finally, the results obtained by all techniques performed were very interesting. The molecular weight calculated for the protein is 61 ± 4 kDa. The study allows us to demonstrate for the first time a polymorphism of at least five members in the human SERPINA3 and a cross-class inhibition for caspase 3 which is approximately 50 %. However, it seemed that it didn’t inhibited so effectively like the bovSERPINA3. As for trypsin and elastase, no inhibition was recorded.