Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Colorimetric detection of metals using HNB provides a simple way to quantify the degree of protein contamination by transition metal ions from IMAC resins. As we established in Ref. 20, Ni²⁺ binds to HNB with 1:1 stoichiometry and the dissociation constant for the Ni-HNB complex changes with pH. However, the complex K_d is in the sub-nM range for all recommended (7-12) pH values. In practical terms, it means that all Ni²⁺ in any tested fractions will bind to HNB as long as no other strong chelating reagents, like EDTA, are present. All these properties together result in linear Ni²⁺ titration curves, which we experimentally observed. In that report²⁰, we also established that the spectral changes due to metal-dye complex formation will be the same over the entire 7-12 pH range. The detection is limited by the minimal reliable absorbance change measurements (10^-4 – 10^-3 OD depending on the spectrophotometer used) corresponding to 2.7-27 nM Ni²⁺. The upper range is limited by the amount of HNB present. In our work, we use ~15 µM, corresponding to ~0.6 OD at 647 nm. However, this can be increased up to 50-80 µM HNB, if needed. Practically, we observed Ni²⁺ contamination levels in chromatographic fractions comparable or higher than the upper limit forcing us to make 10- to 50-fold dilutions of assayed fractions. However, this additional dilution step can increase relative errors while determining the nickel concentration in a fraction.

Though we have not investigated the details, it appeared that binding of other metals used in IMAC resins (Co, Zn, Fe) also has sub-µM dissociation constants and virtually no overlap between dye and dye-metal absorbance spectra at 647 nm, the peak wavelength of free HNB. Therefore, complete metal binding to the dye and the associated spectral changes of the dye can be used for absolute metal determination over the entire recommended pH range.

Execution of the protocol is straightforward and depends most heavily on proper laboratory technique. Modern spectrophotometers have highly linear responses and dynamic ranges of 3-4 orders of magnitude. Consequently, the most likely place for the introduction of error in the method is through the pipetting steps for sample preparation. As described in this text, the method is based on the quantification of metal content based on the difference in the HNB absorbance peak at 647 nm from a free HNB control and samples with HNB complexed with a metal. If care is not taken to accurately pipette the HNB aliquots or the buffer volumes, comparison of the control and sample absorbances at 647 nm becomes a point of error. Similarly, poor pipetting of protein fractions for sample preparation can skew the perceived concentration of metal in a fraction. It is recommended that, due to the sensitivity of the assay, any pipettes being used for analyses where precise quantification is required be calibrated prior to use.

The primary limitations of the method come with the functional pH range of the assay and the presence of strong chelating agents. The assay is best utilized in a pH range from 7-12. Below pH 7, the spectrum of the free HNB dye changes, losing the peak at 647 nm used for quantification²⁰. Above pH 12, many metal hydroxides begin to precipitate, including those of metals commonly found in IMAC resins, making quantification slower and less reproducible. While the alkaline maximum does not pose a significant problem as purification procedures rarely call for such a high pH, the acidic minimum is more likely to be a limiting factor. Since the detection limits for Ni²⁺ and other transition metals are approximately 1000-fold lower than metal contamination levels demonstrated above (Figure 3), the low pH limit for the assay can be circumvented by dilution of the assayed acidic protein fractions in buffers with neutral pH values and sufficiently high buffering capacity. Alternatively, the pH of analyzed fractions can be adjusted or the HNB stock solution can be more strongly buffered to maintain the desired pH after mixing.

If the isolation procedure for the protein being purified requires the use of reagents with known or suspected transition metal chelating properties, a modification of the method would be necessary to allow for proper quantification of leached metals. A standard curve would need to be prepared using the chelating agent used for protein elution and known concentrations of metal standards to accurately quantify the concentration of leached metal in the presence of the chelator. An example of metal quantification in the presence of histidine is available in Kokhan & Marzolf²⁰.

Accurate quantification of metals in biological samples is still largely dependent on the use of analytical techniques and instrumentation, such as AAS and ICP-MS, that remain outside the realm of the typical biochemist^31,32. Bonta et al. have described the simple preparation of biologic samples on common filter paper for analysis by ICP-MS, however, their method still relies on non-standard instrumentation for a biochemist³¹. The method we describe allows for the measurement of metal content in a sample to be taken without additional training on new instrumentation or outsourcing to others. Similar colorimetric protocols have been developed for metal analysis in biological samples³³. However, the method described by Shaymal et al.³³ relies on a fluorescence assay using a commercially unavailable fluorescent probe that gives a higher limit of detection than that in this paper. Considering the relative ease by which the described method can be performed and the recent interest in the development of portable metal detecting protocols for aqueous samples^34,35, it could be easily adapted for field testing of water samples. As a portable test, our method could be modified for use with a portable spectrophotometer for quantification or as a qualitative measure to identify samples for further analysis at a fixed testing location.