Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue
Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue
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DOI：
10.1016/j.ab.2019.113347
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发表时间：
2019-10-01
影响因子：
2.9
通讯作者：
Marzolf, Daniel R.
中科院分区：
文献类型：
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作者：
Kokhan, Oleksandr;Marzolf, Daniel R.
固定化金属亲和层析（IMAC）的广泛使用，快速和有效的重组蛋白质的纯化带来了潜在的有毒过渡元素到常见的实验室使用。然而，很少有研究从亲和树脂，如镍-次氮基三乙酸（Ni-NTA）的金属浸出，可能对生物活性的有害影响。当使用比咪唑更强的还原或螯合洗脱剂时，这是特别重要的。我们提出了一个详细的研究羟基萘酚蓝（HNB）作为一个指标的几个二价金属阳离子，但重点是镍2+，澄清和纠正许多错误和歧义，在旧的文献中，这种染料化合物。该检测方法简单而灵敏，许多金属，特别是Ni 2+、Zn 2+、Cu 2+、Pb 2+、Fe 2+、Co 2+和Al 3+，可以在中性pH和最常用的缓冲液中使用典型生物化学研究实验室中可用的光谱设备在低至1 - 5 -50 nM（1-5 ppb）的浓度下容易地检测和定量。使用这种方法，我们表明，显着量的Ni 2+（高达20 mM）与目标蛋白质（细胞色素bc（1）复合物）共纯化时，组氨酸是用来从Ni-NTA树脂上洗脱。
The widespread use of immobilized metal-affinity chromatography (IMAC) for fast and efficient purification of recombinant proteins has brought potentially toxic transition elements into common laboratory usage. However, there are few studies on the leaching of metal from the affinity resin, such as nickel-nitrilotriacetic acid (Ni-NTA), with possible deleterious impact on the biological activity. This is of particular importance when reducing or chelating eluants stronger than imidazole are used. We present a detailed study of hydroxynaphthol blue (HNB) as an indicator of several divalent metal cations, but with emphasis on Ni2+, clarifying and correcting many errors and ambiguities in the older literature on this dye compound. The assay is simple and sensitive and many metals, notably Ni2+, Zn2+, Cu2+, Pb2+, Fe2+, Co2+, and Al3+, can be readily detected and quantified at concentrations down to 15-50 nM (1-5 ppb) at neutral pH and in most commonly used buffers using spectroscopic equipment available in typical biochemistry research labs. Using this method, we show that significant amounts of Ni2+ (up to 20 mM) are co-purified with a target protein (cytochrome bc(1) complex) when histidine is used to elute from Ni-NTA resin.