ISOLATION AND CHARACTERIZATION OF ALPHA(1)-PROTEASE INHIBITOR FROM CANINE PLASMA

Objective-To improve a previously described purification process by pr oducing a higher yield and purity of alpha(1)-protease inhibitor (alph a(1)-PI) from canine plasma. Animals-Plasma pool from 10 clinically no rmal male dogs. Procedure-Canine alpha(1)-PI was purified by use of am monium sulfate precipitation, ion-exchange chromatography, and 3 affin ity chromatographic procedures: concanavalin A-Sepharose, thiol, and h emoglobin-Sepharose. Characterization was performed by gel electrophor esis, isoelectric focusing, and immunoblot analysis. The N-terminal am ino acid sequence was obtained by use of the Edman degradation method and a gas amino acid sequencer. Results-Canine alpha(1)-PI was purifie d with a yield of approximately 7% and a 54-fold increase in specific inhibitory activity. The inhibitor had a molecular weight of 59,000 an d had 2 major patterns after isoelectric focusing: fast and intermedia te 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 alpha(1)-PI with tho se of sheep, horse, and human alpha(1)-protease inhibitors were 54, 46 , and 41%, respectively. Conclusions-Canine protease inhibitor is anal ogous to the alpha(1)-protease inhibitors of sheep, human beings, and mice in terms of molecular weight, amino acid composition, and inhibit ory activity against trypsin. Although the method described had a yiel d of 7%, the final product retained inhibitory activity and was pure. Clinical Relevance-The availability of pure canine alpha(1)-PI, as wel l as the specific antibodies, will facilitate studies on the fecal exc retion and structural heterogeneity of this protein in dogs with natur ally acquired protein-losing enteropathy.