Enzymatic probe sonication:: Enhancement of protease-catalyzed hydrolysis of selenium bound to proteins in yeast
Enzymatic probe sonication:: Enhancement of protease-catalyzed hydrolysis of selenium bound to proteins in yeast
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DOI：
10.1021/ac034871d
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发表时间：
2004-01-01
影响因子：
7.4
通讯作者：
Cámara, C
中科院分区：
文献类型：
--
作者：
Capelo, JL;Ximénez-Embún, P;Cámara, C
本文描述了两种蛋白水解酶（蛋白酶 XIV 和枯草杆菌蛋白酶）在经超声波探头处理后活性显着增强，及其在生物样品中总硒测定和硒形态分析中的应用。使用 10 mg 酵母加 1 mg 蛋白酶，超声处理时间为 5 秒，从富集酵母中提取总硒，而提取硒代蛋氨酸则需要 30 秒。在这两种情况下，均使用水介质。这一惊人的发现非常重要，因为酶处理过程通常需要在 37 摄氏度下进行长时间的处理。除了这一主要优点之外，不需要控制温度，并且大大降低或抑制了物质相互转化的风险（在不同的超声处理时间后检测到相同的 Se 物质）。此外，提取在水中进行，最大限度地降低了污染风险，且无需进一步调整 pH 值。所提出的新样品处理方法已成功应用于酵母中的硒形态分析，采用色谱分离 (HPLC) 与电感耦合等离子体质谱联用。
This paper describes the dramatic activity enhancement of two proteolytic enzymes (protease XIV and subtilisin) when treated with an ultrasonic probe and their application to total Se determination and Se speciation in biological samples. Total Se extraction from enriched yeast is performed with 10 mg of yeast plus 1 mg of protease with a sonication time of 5 s, whereas 30 s is needed for extracting selenomethionine. In both cases, aqueous media was used. This spectacular finding is important because the enzymatic procedure usually requires a long treatment period at 37 degreesC. In addition to this major advantage, no control temperature is needed and the risk of species interconversion is drastically reduced or inhibited (the same Se species were detected after different sonication times). Moreover, the extraction is performed in water, minimizing contamination risk and without further pH adjustment. The new sample treatment proposed has been successfully applied to selenium speciation in yeast using chromatographic separation (HPLC) coupled to inductively coupled plasma-mass spectrometry.