[en] Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Mathy, Marianne ; Université de Liège - ULiège > Département des sciences de la motricité > Unité de recherche sur l'os et le cartillage (U.R.O.C.)
Bourgeois, E.
Grulke, Sigrid ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Département clinique des animaux de compagnie et des équidés
Deby, Ginette ; Université de Liège - ULiège > Centre de l'oxygène : Recherche et développement (C.O.R.D.)
Caudron, I.
Deby, C.
Lamy, Maurice ; Centre Hospitalier Universitaire de Liège - CHU > Anesthésie et réanimation
Serteyn, Didier ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Anesthésiologie gén. et pathologie chirurg. des grds animaux
Language :
English
Title :
Purification of myeloperoxidase from equine polymorphonuclear leucocytes.
Publication date :
1998
Journal title :
Canadian Journal of Veterinary Research
ISSN :
0830-9000
Publisher :
Canadian Veterinary Medical Association, Ottawa, Canada
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