从有限的血液量中共同纯化马中性粒细胞弹性蛋白酶和髓过氧化物酶的方法。

Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume.

机构信息

Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine B41, University of Liège, Sart Tilman, 4000 Liège, Belgium.

出版信息

Res Vet Sci. 2009 Dec;87(3):358-63. doi: 10.1016/j.rvsc.2009.04.002. Epub 2009 May 5.

DOI:10.1016/j.rvsc.2009.04.002

PMID:19409585

Abstract

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 460 ml 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 34.3 x for elastase (final yield: 340 microg) and MPO (final yield: 130 microg), 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.

摘要

中性粒细胞髓过氧化物酶（MPO）和弹性蛋白酶可在严重炎症性疾病中释放，并导致组织损伤。马酶已从大量血液中被单独纯化。我们描述了从相同的有限血液量中分离这两种酶。通过从 460ml 血液采集物中用 Percoll 梯度离心分离 PMN 来破碎 PMN ，可以提取 MPO 和弹性蛋白酶。通过离子交换色谱相分离 MPO 和弹性蛋白酶，并分别在 Superdex 200 和 75 上通过凝胶过滤色谱进一步纯化。通过特异性酶测定法在收集的级分中鉴定酶。通过电泳验证最终纯度。在该过程中，弹性蛋白酶（最终产率：340μg）和 MPO（最终产率：130μg）的比活性分别提高到 19.92 和 34.3x。结果表明，有可能从同一血液样本中分离出这两种酶，其产率和纯度足以满足对其在炎症性疾病中的作用和相互作用进行未来研究的需要。