Isolation and characterization of α1-protease inhibitor from canine plasma

Tonatiuh Melgarejo, David A. Williams, G. Griffith

Research output: Contribution to journalArticlepeer-review

Abstract

Objective - To improve a previously described purification process by producing a higher yield and purity of α1-protease inhibitor (α1-Pl) from canine plasma. Animals - Plasma pool from 10 clinically normal male dogs. Procedure - Canine α1-Pl 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-Pl 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-Pl 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-Pl, 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.

Original languageEnglish (US)
Pages (from-to)258-263
Number of pages6
JournalAmerican journal of veterinary research
Volume57
Issue number3
DOIs
StatePublished - Mar 1996
Externally publishedYes

ASJC Scopus subject areas

  • General Veterinary

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