Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS Journal of Chromatography B Pub Date : 2024-06-12 DOI:10.1016/j.jchromb.2024.124186

Zhihui Zhao , Zhiyuan Liu , Qiyao Jiang, Xiaoqing Zhang, Wenfu Ma, Dongran Han

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Abstract

Objective

Optimize the extraction process of earthworm fibrinolytic enzyme.

Methods

Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme.

Results

Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods.

Discussion

The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.

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优化高活性蚯蚓纤维蛋白溶解酶提取方法和蛋白质成分分析

方法中国普通蚯蚓经过研磨、盐析、疏水介质层析、硫酸铵沉淀、离子交换层析等一系列纯化过程，获得纯化的蚯蚓纤维蛋白溶解酶。结果以蚯蚓纤溶酶为起始原料，我们发现通过硫酸铵沉淀法提取的纤溶酶比活度为 58 U/mg，明显超过了通过热沉淀法和乙醇沉淀法提取的纤溶酶。经过疏水亲和层析和阴离子交换层析两轮色谱分离后，腰激酶蛋白的比活飙升至9267 U/mg，大大超过了工业化提取方法获得的3178 U/mg比活。高效纯化方法的发现和高活性腰激酶蛋白质成分的鉴定为进一步研究这些蛋白质铺平了道路。

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来源期刊

Journal of Chromatography B 医学-分析化学

CiteScore

5.60

自引率

3.30%

发文量

306

审稿时长

44 days

期刊介绍： The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.