Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Summary

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

Abstract

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

Introduction

Since its inception by Porath and co-workers¹, immobilized metal affinity chromatography (IMAC) has become a method of choice to quickly separate proteins based on their ability to bond with transition metal ions such as Zn²⁺, Ni²⁺, Cu²⁺, and Co²⁺. This is most commonly done via engineered poly-histidine tags and is now one of the most common chromatographic purification techniques for the isolation of recombinant proteins². IMAC has also found applications beyond recombinant protein purification as a way to isolate quinolones, tetracyclines, aminoglycosides, macrolides, and &#....

Protocol

1. Assay component preparation

1. Determine the chromatography fractions to be assayed using optical absorbance at 280 nm or alternative methods of protein quantification to identify the protein enriched fractions.
   NOTE: For this work, we used a diode array UV-Vis spectrophotometer. To increase throughput, a plate reader capable of measuring UV-Vis absorbance can be used.
2. Preparation of necessary assay components
   1. Prepare or obtain 10-100 mM buffer ("Sample Buffer") with a pH between 7 and 12.
      NOTE: Common biochemical buffers such as Tris, HEPES, MOPS, and phosphate at neutral or basic pH are all acceptable for the ....

Results

The spectrum of free HNB at neutral pH (black line) and representative spectra of fractions assayed for Ni²⁺ from the isolation of MSP1E3D1²⁹ are shown in Figure 2. A successful assay series should demonstrate a decreased absorbance at 647 nm compared to the HNB control, which corresponds to the formation of HNB complexes in the presence of a transition metal. A failed assay would be indicated by an increase in absorbance at 647 nm. Alternatively, more than.......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
2xYT broth	Fisher Scientific	BP9743-500	media for E.coli growth
HEPES, free acid	BioBasic	HB0264	alternative buffer
HisPur Ni-NTA resin	Thermo Scientific	88222
Hydroxynaphthol blue disoidum salt	Sigma-Aldrich	219916-5g
Imidazole	Fisher Scientific	O3196-500
Imidazole	BioBasic	IB0277
MOPS, free acid	BioBasic	MB0360	alternative buffer
Sodium chloride	Fisher Scientific	S271-500
Sodium phosphate	Fisher Scientific	S369-500	alternative buffer
Tricine	Gold Bio	T870-100
Tris base	Fisher Scientific	BP152-500
Triton X-100	Sigma-Aldrich	T9284-500