Purification of two equine pepsinogens by use of high-performance liquid chromatography

Abstract

Objectives - To purify and characterize pepsinogens in equine gastric mucosa. Sample Population - Stomachs collected from 2 healthy horses at necropsy. Procedure - After collection, stomachs were placed immediately in ice before storage at -48 C. After slow thawing, the mucosa was scraped off while the tissue was immersed in 0.1 M potassium phosphate (pH 7.4) at 4 C, then was homogenized. The filtered extract was subjected to anion-exchange chromatography. Fractions that were found to contain pepsin or pepsinogen were further chromatographed. Individual fractions were tested for pepsinogen or pepsin content by monitoring proteolytic activity at pH 2 and 3, respectively. Fractions from all columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to confirm molecular weight of pepsinogens and pepsin. Results - Two pepsinogens and at least 1 pepsin were purified from equine gastric mucosa. Conclusions - On the basis of molecular mass, equine gastric mucosa contains 2 pepsinogens. Clinical Relevance - Cellular immune response-based and serologic tests are useful tools for seeking evidence of paratuberculosis. Each technique detects different types of immune-reacting sheep, and their simultaneous use may detect most infected animals. (Am J Vet Res 1999;60:123-127).