Isolation and characterization of alpha 1-protease inhibitor from canine plasma.
OBJECTIVE
To improve a previously described purification process by producing a higher yield and purity of alpha 1-protease inhibitor (alpha 1-PI) from canine plasma.


METHODS
Plasma pool from 10 clinically normal male dogs.


METHODS
Canine alpha 1-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 alpha 1-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 alpha 1-PI with those of sheep, horse, and human alpha 1-protease inhibitors were 54, 46, and 41%, respectively.


CONCLUSIONS
Canine protease inhibitor is analogous to the alpha 1-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.


CONCLUSIONS
The availability of pure canine alpha 1-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.
Melgarejo
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Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN 47907-1248, USA.
Williams
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DA
Griffith
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006579:Heterozygote; D006720:Homozygote; D006736:Horses; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D000906:Antibodies; D000515:alpha 1-Antitrypsin