Sample preparation for metalloprotein analysis: A case study using horse chestnuts
Sample preparation for metalloprotein analysis: A case study using horse chestnuts
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DOI：
10.1016/j.talanta.2006.08.039
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发表时间：
2007-03-30
期刊：
影响因子：
6.1
通讯作者：
Zezzi Arruda, Marco Aurelio
中科院分区：
文献类型：
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作者：
de Magalhaes, Cristiana Schmidt;Zezzi Arruda, Marco Aurelio
本文研究了从马栗中提取蛋白质和金属蛋白的不同工艺。在Natura进行了测试。每次提取后，测定总蛋白，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质后，用同步辐射X射线荧光(SRXRF)对属于蛋白质结构的金属进行定位。通过绘制蛋白质条带(约33 kDa和23.7 kDa)中的元素(铬、铁和锰)，用原子吸收光谱仪(ET AAS)测定其浓度，并将研磨和超声相结合的方法用于蛋白质提取，取得了良好的效果。然而，这种策略不适合保存蛋白质结构中的金属离子。事实上，与在水中搅拌样品(作为参考)相比，使用该方法对锰的浓度降低了42%。另一方面，当使用研磨和搅拌提取缓冲液时，与参考方法相比，锰的浓度增加了530%。这一结果表明金属鉴定与蛋白质中的测定是一致的，以及提取程序(即样品制备步骤)对保存蛋白质结构中的金属的影响很大。(C)2006爱思唯尔B.V.保留所有权利。
In the present work, I I different procedures for protein and metalloprotein extraction from horse chestnuts (Aescullus hippocastanum L..) in natura were tested. After each extraction, total protein was determined and, after protein separation through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), those metals belonging to the protein structure were mapped by synchrotron radiation X-ray fluorescence (SRXRF). After mapping the elements (Cr, Fe and Mn) in the protein bands (ca. 33 and 23.7 kDa), their concentrations were determined using atomic absorption spectrometry (ET AAS).Good results were obtained for protein extraction using a combination of grinding and sonication. However, this strategy was not suitable to preserve metal ions in the protein structure. In fact, there was 42% decrease on Mn concentration using this procedure, compared to that performed with sample agitation in water (taken as reference). On the other hand, when grinding and agitation with an extracting buffer was used, there was a 530% increase of Mn concentration, when compared to the reference procedure.These results indicate agreement between metal identification and determination in proteins as well as the great influence of the extraction procedure (i.e., the sample preparation step) for preserving metals in the protein structures. (c) 2006 Elsevier B.V. All rights reserved.