[en] Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities. We describe the isolation of both enzymes from a same limited blood volume. Both MPO and elastase were extracted by crushing PMN isolated by centrifugation on a percoll-gradient from a 460ml blood collection. MPO and elastase were separated by an ionic exchange chromatography phase and further purified by gel filtration chromatography on Superdex 200 and 75, respectively. Enzymes were identified in the collected fractions by specific enzymatic assays. The final purity was verified by electrophoresis. Specific activity was improved to 19.92 and 34.3x for elastase (final yield: 340mug) and MPO (final yield: 130mug), respectively, during the procedure. Results show the possibility of isolating both enzymes from the same blood sample with a sufficient yield and purity for future studies on their implication and interaction during inflammatory diseases.
Centre de l’Oxygène, Recherche et Développement - CORD
Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (Communauté française de Belgique) - FRIA