College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, The People's Republic of China.
Biometals. 2014 Apr;27(2):265-76. doi: 10.1007/s10534-014-9707-x. Epub 2014 Feb 18.
Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.
铜（Cu）是植物生长和发育所必需的一种必需的微量元素。然而，由于不可避免地与蛋白质结合，过量的 Cu 会使蛋白质失活并扰乱其结构。为了更好地理解植物中铜毒性和耐受性的机制，我们开发了一种新的固定金属亲和色谱（IMAC）方法，用于分离和分离暴露于过量 Cu 的水稻幼苗根中提取的 Cu 结合蛋白。在我们的方法中，IDA-Sepharose 或 EDDS-Sepharose 柱（称为预色谱）和 Cu-IDA-Sepharose 柱（称为 Cu-IMAC）串联连接。也就是说，蛋白质样品先用 IDA-Sepharose 柱预色谱分离，以去除金属离子，然后将蛋白质溶液流入 Cu-IMAC 柱，在体外富集 Cu 结合蛋白。与对照（未经任何预色谱的 Cu-IMAC）相比，IDA-Sepharose 预色谱法显著提高了 Cu-IMAC 结合蛋白的产量、二维电泳（2-DE）凝胶上的蛋白质斑点数量和 40 个蛋白质斑点的丰度。从 2-DE 凝胶中随机选择 13 个蛋白质斑点，并用 MALDI-TOF-TOF MS 鉴定了 11 个蛋白质。这些假定的 Cu 结合蛋白包括参与抗氧化防御、碳水化合物代谢、核酸代谢、蛋白质折叠和稳定、蛋白质运输和细胞壁合成的蛋白。10 种蛋白质含有 Smith 等人报道的 9 种假定金属结合基序中的一种或多种（J Proteome Res 3:834-840, 2004），7 种蛋白质含有 Kung 等人报道的前 6 种基序中的一种或两种（Proteomics 6:2746-2758, 2006）。结果表明，通过 IDA-Sepharose 预色谱从样品中去除金属离子，可以更有效地富集与 Cu-IMAC 特异性结合的蛋白质。需要进一步研究这些蛋白质在体内的金属结合特性以及 Cu 离子与蛋白质生物活性之间的关系，以充分理解植物中 Cu 耐受性和毒性的机制。
