Name: quantification of metal leaching in immobilized metal affinity chromatography
Uploaded: 2020-01-17T16:00:00.000+00:00
Duration: 5 min 35 s
Description: Watch this Scientific Journal Video about Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography at JoVE.com

This material is based upon work supported by the National Science Foundation under Grant MCB-1817448 and by an award from the Thomas F. and Kate Miller Jeffress Memorial Trust, Bank of America, Trustee and specified donor Hazel Thorpe Carman and George Gay Carman Trust.

1. Assay component preparation
<ol>
	<li>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.</li>
	<li>Preparation of necessary assay components
	<ol>
		<li>Prepare or obtain 10-100 mM buffer (&#34;Sample Buffer&#34;) 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 assay. Tricine and histidine can be used but will require calibration curves as they both substantially chelate metal ions. An example of calibration for histidine is shown in reference20.</li>
		<li>Prepare a 12% w/v (20-fold concentrated) solution of hydroxynaphthol blue (HNB) dispersion in the Sample Buffer using 120 mg of HNB reagent for each milliliter of stock solution prepared. 
		CAUTION: Exposure of HNB to the eye can cause serious damage and irritation. Eye protection should be used when handling HNB and hands should be washed thoroughly after handling. 
		NOTE: HNB is sold as a dispersion on KCl by major suppliers of scientific reagents. As such, actual concentration in solution will vary from different manufactures, batches, and where in the bottle the HNB dispersion is taken from. Ideally, an absorbance between 0.5-0.8 at 647 nm after 20-fold dilution of the stock should be achieved.</li>
	</ol>
	</li>
</ol>
2. Sample preparation and measurement
<ol>
	<li>Preparation of the spectrophotometer for data collection
	<ol>
		<li>Turn on and warm up the UV-Vis spectrophotometer. Set the spectrophotometer to collect data at 647 nm. 
		NOTE: If the spectrophotometer allows, additionally collect data at 850 nm, or some other wavelength without significant changes related to the dye-metal and dye spectra, to be used for a baseline correction.</li>
		<li>Blank the spectrophotometer using the Sample Buffer. 
		NOTE: Quartz or disposable plastic cuvettes may be used. Quartz cuvettes are preferred for quantitative analysis as they will allow higher accuracy and precision over disposable plastic cuvettes. However, plastic cuvettes block UV light, which may be present in the measuring beams of some diode-array spectrophotometers. Exposure of HNB to intense UV light causes notable dye degradation and an unwanted slow absorbance decrease that can be confused with slow metal binding (for example, see Figure 1 in Supporting Information of Reference 20).</li>
	</ol>
	</li>
	<li>Preparation and absorbance measurement of control
	<ol>
		<li>Prepare a control solution containing 50 &#956;L of HNB stock per milliliter of total assay volume. To ensure good mixing of all samples, pipette the small volumes first then add the Sample Buffer followed by mixing by pipetting. Diluted HNB solution should be prepared fresh but HNB stocks can be stored at 4 &#176;C and shielded from light for weeks without significant degradation.</li>
		<li>Allow the control to incubate for a minimum of 3 min at room temperature. 
		NOTE: A longer incubation time may be necessary for samples at alkaline pH or in the presence of phosphate due to formation of poorly soluble metal complexes resulting in slower equilibration.</li>
		<li>Measure and record the absorbance at 647 nm for the control sample.</li>
	</ol>
	</li>
	<li>Preparation and absorbance measurement of samples
	<ol>
		<li>Prepare the assay samples by mixing 50 &#956;L of HNB stock with 950 &#181;L of appropriately diluted protein fractions with the Sample Buffer. 
		NOTE: Since metal contamination levels reported in literature vary by a factor of more than 1000 depending on elution conditions16,20, it may be necessary to try a few dilutions of the assayed protein fractions with the Sample Buffer (see step 1.2.1 above) to achieve absorbance changes within the dynamic range of the assay. 
		CAUTION: Nickel and other metals used in IMAC are known skin irritant, suspected of being carcinogenic, and are capable of damaging the kidneys and blood after prolonged exposure. Gloves and eye protection should be used when handling protein samples prepared with IMAC.</li>
		<li>Allow the sample to incubate for a minimum of 3 min at room temperature and measure absorbances at 647 nm. 
		NOTE: The limiting step of the assay in terms of time invested is the incubation step. The data for this paper was collected using a single quartz cuvette that was carefully washed between each sample. Even with the added washing time and preparation of the HNB stock, data collection for 14 samples and the control took approximately an hour and a half and as such, the protocol can be easily completed without the need for interruption.</li>
		<li>Repeat steps 2.3.1 and 2.3.2 for each fraction that will be measured. 
		NOTE: If several cuvettes will be used for multiple samples, samples should be prepared in a way that allows for comparable incubation time and exposure to ambient light.</li>
	</ol>
	</li>
</ol>
3. Metal quantification
<ol>
	<li>Determining the concentration of metal in each sample
	<ol>
		<li>Find the difference of each sample absorbance at 647 nm from the HNB control.</li>
		<li>Determine the metal concentration (in &#181;M) using the formula below: 
		 
		<img alt="Equation 1" src="/files/ftp_upload/60690/60690equfig01.jpg" /> 
		 
		where DF is the dilution factor for the assay fraction, &#916;Abs647 is the absorbance change at 647 nm, 3.65x10-2 represents the extinction coefficient of HNB (&#949;=36.5 mM-1&#183;cm-1 see Reference20 for more details) and l is cuvette&#39;s optical path in cm.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 Kd 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 &#8211; 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 &#181;M, corresponding to ~0.6 OD at 647 nm. However, this can be increased up to 50-80 &#181;M 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-&#181;M 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 &#38; 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 biochemist31,32. 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 samples34,35, 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.

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 &#946;-lactams for food sample analysis3 and as a step in identifying blood-serum protein markers for liver and pancreatic cancers4,5. Not surprisingly, IMAC has also become a method of choice for the isolation of a number of native bioenergetics enzymes6,7,8,9,10. However, successful implementation of these purification methods for studies on enzymatically active bioenergetic proteins is dependent on the presence of negligible levels of metal cations leached from the column matrix into the eluate. The divalent metal cations commonly used in IMAC have known pathologic biological significance, even at low concentrations11,12. The physiological effect of these metals is most pronounced in bioenergetic systems, where they can prove lethal as inhibitors of cellular respiration or photosynthesis13,14,15. Similar issues are unavoidable for the majority of protein classes where residual contaminant metals can interfere with a protein&#39;s biological functions or characterization with biochemical and biophysical techniques.
While the levels of metal contamination under oxidizing conditions and using imidazole as an eluant are typically low16, protein isolations performed in the presence of cysteine reducing agents (DTT, &#946;-mercaptoethanol, etc.) or with stronger chelators like histidine17,18 or ethylenediaminetetraacetic acid (EDTA) result in much higher levels of metal contamination19,20. Similarly, since metal ions in IMAC resins are frequently coordinated by carboxylic groups, protein elutions performed under acidic conditions are also likely to have much higher levels of metal contamination. Metal content in solutions can be assessed using atomic absorption spectroscopy (AAS) and inductively coupled plasma-mass spectrometry (ICP-MS) down to a limit of detection in the ppb-ppt range21,22,23,24. Unfortunately, AAS and ICP-MS are not realistic means for detection in a traditional biochemistry lab as those methods would require access to specialized equipment and training.
Previous work by Brittain25,26 investigated the use of hydroxynaphthol blue (HNB) as a way to identify the presence of transition metals in solution. However, there were several internal contradictions in the data20 and those works failed to offer an adequate protocol. Studies by Temel et al.27 and Ferreira et al.28 expanded on Brittain&#39;s work with HNB as a potential metal indicator. However, Temel developed a protocol that makes use of AAS for sample analysis, using HNB only as a chelating agent. Ferreira&#39;s study used the change in the HNB absorbance spectra at 563 nm, a region of the free-dye HNB spectra that overlaps heavily with the spectra of HNB-metal complexes at pH 5.7, making the assay sensitivity fairly low as well as resulting in relatively weak metal binding affinity20. To address issues in our own lab with Ni2+ leaching from IMAC, we have expanded the work done by Brittain25,26 and Ferreria28 to develop an easy assay capable of detecting nanomolar levels of several transition metals. We showed that HNB binds nickel and other common for IMAC metals with sub-nanomolar binding affinities and form 1:1 complex over a wide range of pH values20. The assay reported here is based on these findings and utilizes absorbance changes in the HNB spectrum at 647 nm for metal quantification. The assay can be performed in the physiological pH range using common buffers and instrumentation found in a typical biochemistry lab by using colorimetric detection and quantification of metal-dye complexes and the associated change in absorbance of the free-dye when it binds to metal.

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

quantification of metal leaching in immobilized metal affinity chromatography

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.

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 90% decrease from initial absorbance at 647 nm would indicate too high metal content and a need to assay more diluted fractions. An assay with no absorbance changes from the free HNB control does not necessarily indicate a failure. It is possible that samples contain essentially no leached metals. However, this is unlikely and any sample showing no absorbance change should be prepared and measured again, preferably with less dilution, to confirm the result. In total, most failures to observe the expected absorbance change can be attributed to improper pipetting during sample preparation, an inadequate incubation time prior to measurement, or pH values outside the recommended 7-12 range.
To demonstrate the application of this assay, we analyzed 2 His-tag membrane scaffold proteins MSP1E3D1 (isolated as in Denisov, I. G. et al.29), MSP2N2 (isolated as in Grinkova, Y. V.30), and a novel 3-heme c-type cytochrome GSU0105 from Geobacter sulfurreducens, which was recombinantly expressed in E.coli and eluted with 500 mM imidazole. The elution profiles of Ni2+ from a Ni-NTA resin column (see Table of Materials) and the associated protein elution profiles for these 3 proteins are shown in Figure 3. Any protein will have a unique nickel elution that may or may not align with the protein elution profile as measured at 280 nm. For example, Figure 3C shows that the protein and Ni2+ content of each fraction for GSU0105 are significantly shifted from one another while the fractions for MSP1E3D1 and MSP2N2 (Figure 3A,B) that contain the most protein also have the highest Ni2+ content. Figure 3A,B also illustrate that metal content may not be evenly distributed among fractions collected using IMAC. Depending on column packing, the composition of the elution buffer, pumping equipment and conditions, it is possible to have metal elute in consecutive fractions at very different concentrations independent of the protein content of those fractions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60690/60690fig1a.jpg" /> 
Figure 1: Structure of hydroxynaphthol blue (HNB). In the functional pH range of the assay, all sulfonate groups and one of the hydroxyl groups are ionized. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60690/60690fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60690/60690fig2a.jpg" /> 
Figure 2: Absorbance spectra for selected MSP1E3D1 fractions and HNB. The relative absorbance of three fractions of MSP1E3D1 (colors) compared to an HNB control (black thick line) are shown. Samples were prepared in 20 mM Tris, pH 7.5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60690/60690fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60690/60690fig3a.jpg" /> 
Figure 3: Ni2+ quantification for 3 representative His-tag proteins. Protein and Ni2+ elution profiles for (A) MSP1E3D1, (B) MSP2N2, and (C) GSU0105. Protein elution was performed using 300 mM, 300 mM, and 500 mM imidazole, respectively. Ni2+ quantification was performed in 20 mM Tris, pH 7.5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60690/60690fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography at JoVE.com

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

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.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

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7.2K Views.  James Madison University. Onze methode is significant, omdat het de gemiddelde biochemicus in staat stelt om snel te bepalen of monsters geïsoleerd met behulp van geïmmobiliseerde metaal affiniteit chromatografie zijn besmet met overgang metalen. Het belangrijkste voordeel is het gemak waarmee de test kan worden uitgevoerd. De test maakt gebruik van instrumentatie en technieken gemeenschappelijk voor de meeste biochemie laboratoria, en het kan gemakkelijk en snel worden geïmplementeerd.Terwijl in dit artikel...

Video: Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Environment

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Engineering

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Biochemistry

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (&lt;2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6&#8729;H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested.

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Bioengineering

Food Sample Homogenization and Digestion for Multi-Element Analysis

Preparation of Food Samples Using Homogenization and Microwave-Assisted Wet Acid Digestion for Multi-Element Determination with ICP-MS

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid digestion. Special care is required during sample handling in the preparation stage to eliminate or minimize potential contamination and analyte loss. Homogenization is a process that simultaneously reduces particle size and uniformly distributes sample components. Following homogenization, the sample undergoes acid digestion, wherein it is digested with acids and auxiliary chemicals at elevated temperatures, transforming solid samples into a liquid state. In this process, metals in the original sample react with acids to form water-soluble salts. Samples prepared through acid digestion are suitable for elemental analysis using techniques such as inductively coupled plasma mass spectrometry, inductively coupled plasma optical emission spectroscopy, atomic absorption spectroscopy, electrochemical methods, and other analytical techniques. This work details the preparation of food samples for multi-element determination using inductively coupled plasma mass spectrometry. The step-by-step procedure involves the homogenization process using a laboratory-sized mixer with ceramic blades, followed by acid digestion in closed vessels using microwave-assisted wet acid digestion. A mixture of 5.0 mL of 68 wt% HNO3 and 1.0 mL of 30 wt% H2O2 serves as an auxiliary reagent. This guide provides an explanation of the processes involved in both stages.

Author Spotlight: Technologies and Challenges in Elemental Analysis of Food Samples

The presented protocol describes sample homogenization with a laboratory mixer, acid digestion of food samples using a mixture of 68 wt% HNO3 and 30 wt% H2O2&#160;via microwave-assisted wet acid digestion, and multi-element determination performed with inductively coupled plasma mass spectrometry.

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
</div>
<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
</div>
<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
</div>
<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

</div>
<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
</div>
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Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
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Size Exclusion and Affinity Chromatography

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

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Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

Source: Nachbar M. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57749">Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis</a>. J. Vis. Exp. (2018).
This video describes the method to analyze protein-ligand interaction by mobility-shift affinity capillary electrophoresis. This method can be used for characterizing the binding behavior of a protein with various charged ligands.

Source: Nachbar M. et al., Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

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