Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue

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 bc₁ complex) when histidine is used to elute from Ni-NTA resin.