Department of Chemistry, Texas A&M University, College Station, TX, 77843-3255, USA.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, 77843, USA.
J Biol Inorg Chem. 2021 Jun;26(4):479-494. doi: 10.1007/s00775-021-01864-w. Epub 2021 May 8.
Labile low-molecular-mass (LMM) transition metal complexes play essential roles in metal ion trafficking, regulation, and signalling in biological systems, yet their chemical identities remain largely unknown due to their rapid ligand-exchange rates and weak M-L bonds. Here, an Escherichia coli cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of the metal ions from these complexes with a SEC column was minimized by pre-loading the column with ZnSO and then monitoring Zn and other metals by inductively coupled plasma mass spectrometry (ICP-MS) when investigating cytosolic ultrafiltration flow-through-solutions (FTSs). Endogenous cytosolic salts suppressed ESI-MS signals, making the detection of metal complexes difficult. FTSs contained ca. 80 µM Fe, 15 µM Ni, 13 µM Zn, 10 µM Cu, and 1.4 µM Mn (after correcting for dilution during cytosol isolation). FTSs exhibited 2-5 Fe, at least 2 Ni, 2-5 Zn, 2-4 Cu, and at least 2 Mn species with apparent masses between 300 and 5000 Da. Fe(ATP), Fe(GSH), and Zn(GSH) standards were passed through the column to assess their presence in FTS. Major LMM sulfur- and phosphorus-containing species were identified. These included reduced and oxidized glutathione, methionine, cysteine, orthophosphate, and common mono- and di-nucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the labile metal pools in E. coli is sensitive to the concentration of nutrient metals.
不稳定的低分子量（LMM）过渡金属配合物在生物系统中的金属离子运输、调节和信号传递中发挥着重要作用，但由于其快速的配体交换率和较弱的 M-L 键，其化学性质在很大程度上仍然未知。在这里，开发了一种不含去污剂、强配位缓冲液和 EDTA 的大肠杆菌胞质溶胶分离程序。通过用 ZnSO4 预加载色谱柱，然后在研究胞质超滤液（FTS）时通过电感耦合等离子体质谱法（ICP-MS）监测 Zn 和其他金属，最大限度地减少了这些配合物中的金属离子与 SEC 柱的相互作用。内源性胞质盐抑制了 ESI-MS 信号，使得金属配合物的检测变得困难。FTS 中含有约 80µM Fe、15µM Ni、13µM Zn、10µM Cu 和 1.4µM Mn（在胞质溶胶分离过程中稀释后校正）。FTS 显示 2-5 Fe、至少 2 Ni、2-5 Zn、2-4 Cu 和至少 2 Mn 种，表观质量在 300 至 5000 Da 之间。将 Fe(ATP)、Fe(GSH) 和 Zn(GSH) 标准品通过色谱柱，以评估它们在 FTS 中的存在。鉴定出主要的 LMM 含硫和含磷物质。这些物质包括还原和氧化型谷胱甘肽、甲硫氨酸、半胱氨酸、正磷酸盐以及常见的单核苷酸和二核苷酸，如 ATP、ADP、AMP 和 NADH。在补充了这些金属盐的培养基中生长的细胞的 FTS 显示出补充金属的峰强度增加，表明大肠杆菌中不稳定金属池的大小对营养金属的浓度敏感。
