Isolation and characterization of α1-protease inhibitor from canine plasma

Abstract

Abstract Objective To improve a previously described purification process by producing a higher yield and purity of α1-protease inhibitor (α1-PI) from canine plasma. Animals Plasma pool from 10 clinically normal male dogs. Procedure Canine α1-PI was purified by use of ammonium sulfate precipitation, ion-exchange chromatography, and 3 affinity chromatographic procedures: concanavalin A-Sepharose, thiol, and hemoglobin-Sepharose. Characterization was performed by gel electrophoresis, isoelectric focusing, and immunoblot analysis. The N-terminal amino acid sequence was obtained by use of the Edman degradation method and a gas amino acid sequencer. Results Canine α1-PI was purified with a yield of approximately 7% and a 54-fold increase in specific inhibitory activity. The inhibitor had a molecular weight of 59,000 and had 2 major patterns after isoelectric focusing: fast and intermediate in homozygous and/or heterozygous forms. Edman degradation revealed glutamic acid as the starting amino acid from the N-terminal sequence. Homologies of the N-terminal sequence of canine α1-PI with those of sheep, horse, and human α1-protease inhibitors were 54, 46, and 41 %, respectively. Conclusions Canine protease inhibitor is analogous to the α1-protease inhibitors of sheep, human beings, and mice in terms of molecular weight, amino acid composition, and inhibitory activity against trypsin. Although the method described had a yield of 7%, the final product retained inhibitory activity and was pure. Clinical Relevance The availability of pure canine α1-PI, as well as the specific antibodies, will facilitate studies on the fecal excretion and structural heterogeneity of this protein in dogs with naturally acquired protein-losing enteropathy.(Am J Vet Res 1996; 57:258-263)