Our method is significant, as it allows the average biochemist to quickly determine if samples isolated using immobilized metal affinity chromatography are contaminated with transition metals. The main advantage is the ease by which the assay can be performed. The assay uses instrumentation and techniques common to most biochemistry labs, and it can be easily and quickly implemented.
While in this article we apply this method for samples isolated with a nickel-NTA column, the method can be used with any other metal affinity resin. First time users should try this method with a nickel stock solution of known concentration. This will familiarize them with the workflow, sample colors, and resins shown in spectral changes.
Demonstrating the procedure will be Cole Swain, a technician from my laboratory. To begin, turn on and warm up the UV-Vis spectrophotometer. Determine the chromatography fractions to be assayed using a diode array UV-Vis spectrophotometer with optical absorbance at 280 nanometers to quantify the protein.
Obtain 10 to 100 millimolar sample buffer with a PH between 7 and 12, such as Tris, HEPES, MOPS, and phosphate buffer. Prepare a 12%weight by volume solution of HNB dispersion in the sample buffer using 120 milligrams of HNB reagent for each milliliter of stock solution prepared. Set the spectrophotometer to collect data at 647 nanometers.
Use a quartz cuvette filled with the sample buffer to blank the spectrophotometer. Prepare a control solution in a cuvette containing 50 microliters of HNB stock per milliliter of total assay volume. Allow the control to incubate for a minimum of three minutes at room temperature.
Measure and record the absorbance at 647 nanometers for the control sample. Prepare the assay samples by mixing 150 microliters of HNB stock with 2850 microliters of appropriately diluted protein fractions with the sample buffer. Allow the sample to incubate for a minimum of three minutes at room temperature.
Repeat the spectrum recording for each fraction to be measured. In the case where insufficient dilution is obtained, the entire spectral band at 647 nanometers is gone. While with too-strong dilution, the sample is undistinguishable from the control.
To determine the concentration of metal in each sample, first find the difference of each sample absorbance at 647 nanometers from the HNB control. Determine the metal concentration in micromolar using the formula where DF is the dilution factor for the assay fraction, delta A-B-S 647 is the absorbance change at 647 nanometers, 3.65 times 10 to the negative 2 represents the extinction coefficient of HNB, and l is cuvette's optical path in centimeters. In this study, the spectrum of free HNB at neutral PH in representative spectra of fractions assayed for nickel ion from the isolation of MSP1E3D1 are shown here.
A decreased absorbance at 647 nanometers compared to the HNB control was observed, which corresponded to the formation of HNB complexes in the presence of a transition metal. To demonstrate the application of this assay, two his-tagged membrane scaffold proteins, MSP1E3D1, MSP2N2, and a novel, three-heme, c-type cytochrome GSU0105, from geobacter sulfurreducens, were analyzed. The protein and nickel ion content of each fraction for GSU0105 were significantly shifted from one another, while the fractions for MSP1E3D1 and MSP2N2 that contained the most protein also had the highest nickel content.
It is also illustrated that the metal content may not be evenly distributed among fractions collected using a mobilized metal affinity chromatography. It's most important to adequately and consistently mix the blank in all samples and allow for equal incubation times for the blank in all samples. Protein fractions of special interest could be further analyzed with atomic absorption spectrometry or ICPMS to confirm metal contamination and to test for chelated or strongly bound protein metal ions.
Besides transition metal leaching detection, this technique can be used to measure binding affinities in transition metal ions to proteins. HNB and any nickel present in samples are irritants to the eyes and skin respectively. Standard PPE including gloves and eye protection should be used during the method.
