Affinity Electrophoresis Research Articles

Assessing Target of Rapamycin (TOR) activity in the diatom Phaeodactylum tricornutum using commercially available materials

Target of rapamycin (TOR) is a conserved protein kinase that regulates the balance between catabolic and anabolic processes in response to nutrient availability. Although the central role of TOR kinase in nutrient stress responses is well-recognized, little is known about the molecular basis of TOR signaling in ecologically important secondary algae with plastids of red algal origin, such as diatoms, as assessing in vivo TOR kinase activity is a difficult task. To assess TOR kinase activity, the phosphorylation status of downstream components, such as ribosomal protein S6 (RPS6), must be measured. Unlike for model organisms, an antibody that detects phosphorylated (P-) RPS6 in diatoms is not commercially available. Therefore, we developed a convenient method in which P-RPS6 and non-P-RPS6 were detected via Phos-tag affinity electrophoresis and immunoblotting with a commercial antibody that cross-reacts with RPS6 (both P- and non-P-RPS6) in the diatom, Phaeodactylum tricornutum. Using this Phos-tag-based method, we observed a dose-dependent decrease in the P-RPS6/total RPS6 ratio in P. tricornutum cells treated with the TOR kinase inhibitor, AZD-8055. We also observed a reduction in the P-RPS6/total RPS6 ratio during the nitrogen-deficient culture of P. tricornutum, which indicated the inactivation of TOR kinase in response to nitrogen deficiency. Finally, we demonstrated the potential application of the Phos-tag-based method to other ecologically, evolutionarily, and industrially important secondary algae, such as Nannochloropsis oceanica (Stramenopiles), the haptophyte Tisochrysis lutea, and Euglena gracilis (Euglenid). As all experimental materials are commercially available, the Phos-tag-based method can be used to promote studies on TOR in diverse algae in different contexts.

Determination of physicochemical parameters of (bio)molecules and (bio)particles by capillary electromigration methods.

The review provides an overview of recent developments and applications of capillary electromigration (CE) methods for the determination of important physicochemical parameters of various (bio)molecules and (bio)particles. These parameters include actual and limiting (absolute) ionic mobilities, effective electrophoretic mobilities, effective charges, isoelectric points, electrokinetic potentials, hydrodynamic radii, diffusion coefficients, relative molecular masses, acidity (ionization) constants, binding constants and stoichiometry of (bio)molecular complexes, changes of Gibbs free energy, enthalpy and entropy and rate constants of chemical reactions and interactions, retention factors and partition and distribution coefficients. For the determination of these parameters, the following CE methods are employed: zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography. In the individual sections, the procedures for the determination of the above parameters by the particular CE methods are described.

SYNERGISTIC ANTI-TUMOUR EFFECT OF DOXORUBICIN-HYDROCHLORIDE WITH CRM197, AN INHIBITOR OF HB-EGF, IN SQUAMOUS-CELL CARCINOMA

Recent studies have shown that cross reacting material (CRM197), a nontoxic variant of diphtheria toxin, may play an important role in treating cancers with poor prognoses. Doxorubicin-hydrochloride (DOX) is an antineoplastic prescription medicine for the treatment of certain types of cancer. Considering that CRM197 is a known carrier in targeted delivery, CRM197-DOX complexes might be a step towards targeted therapy and reduced overall toxicity. Aim. This study aims to explore the potential to inhibit the growth of tumour cells. To achieve that goal, we evaluated the usage of CRM197-DOX complexes in squamous carcinoma cell line A431 and compared it with the effect on other immortalised cell lines. The methods used in this research include derivation and purification of recombinant CRM197 through immobilized-metal affinity chromatography and electrophoresis in a denaturing polyacrylamide gel. DOX-loaded CRM197 complexes were formed by the addition of a 5 μM solution of doxorubicin to 0.5 μg of protein with further dialysis in PBS. The cell viability assay was conducted using free CRM197 at different concentrations, DOX, CRM197 with DOX, as well as DOX-loaded CRM197. DOX-loaded CRM197 along with CRM197 with the addition of DOX showed significant differences in cell viability compared to control wells. CRM197- Results. DOX complexes have an evident inhibitory effect on epidermoid carcinoma cell growth and can be used as treatment against epithelial tumours, with CRM197 as a promising carrier for targeted drug delivery. Conclusions. CRM197-DOX complexes show evident inhibition of epidermoid carcinoma cell growth and can be used as treatment against epithelial tumours, especially those overexpressing the proHB-EGF and its receptors, EGFR1 and EGFR4, with CRM197 as a promising carrier for targeted drug delivery.

Liquid Chromatography and Capillary Electrophoresis Analysis of 2AA-Labeled O-Glycans.

To reveal O-glycan structures in mucins, it is necessary to release covalently bound O-glycans from the polypeptide backbone and derivatize to a form suitable for structural analysis. Various derivatization methods can now be applied in the analysis of O-glycans following the development of O-glycan release methods. Among the many derivatization methods available, we prefer to use fluorescent labeling with 2-aminobenzoic acid (anthranilic acid, 2AA). 2AA-labeled O-glycans can be detected with high sensitivity using liquid chromatography fluorescence detection (LC-FD) analysis because of the strong fluorescence. In addition, as 2AA has a carboxyl group that carries a negative charge, 2AA-labeled O-glycans can be analyzed with high sensitivity in negative ion mode mass spectrometry. Furthermore, because the negative charge of 2AA provides a driving force for electrophoresis, 2AA-labeled O-glycans can be analyzed using capillary electrophoresis (CE) and capillary affinity electrophoresis. High detection sensitivity and versatility are key advantages of the 2AA-labeling method. Here we present our preferred LC-FD and CE analytical methods for 2AA-labeled O-glycans.

Recent developments in capillary and microchip electroseparations of peptides (2021-mid-2023).

This review article brings a comprehensive survey of developments and applications of high-performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical characterization of peptides in the period from 2021 up to ca. the middle of 2023. Progress in the study of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, adsorption suppression, electroosmotic flow regulation, and detection, are presented. New developments in the particular capillary electromigration methods are demonstrated, and several types of their applications are reported. They cover qualitative and quantitative analysis of synthetic or isolated peptides and determination of peptides in complex biomatrices, peptide profiling of biofluids and tissues, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They include also amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical characterization of peptides and their interactions with other (bio)molecules by the above CE methods are described.

A Novel Approach for Glycosylated Hemoglobin Testing Using Microchip Affinity Electrophoresis.

Effective management of diabetes largely benefits from early diagnosis followed by intensive long-term regulation of blood glucose. The levels of glycohemoglobin (HbA1 and HbA1c) have been used as standard biomarkers to assess long-term blood glucose concentrations for diabetes diagnosis and management. Gold standard laboratory methods for HbA1 and HbA1c testing are often costly and not widely available. Moreover, currently available point-of-care (POC) immunoassay-based glycohemoglobin tests may produce inaccurate test results for patients with co-existing diseases such as hemoglobin disorders and anemia. Here, we report a POC platform, HemeChip-GHb, for quantitative HbA1 detection leveraging paper-based affinity electrophoresis. We describe the design and development of the HemeChip-GHb test. Feasibility and accuracy of the HemeChip-GHb system were demonstrated by testing blood samples collected from healthy donors, patients with prediabetes, and patients with diabetes. HbA1 levels measured with HemeChip-GHb show 0.96 correlation to the levels reported from the clinical standard HPLC tests, and with a bias of -0.72% based on Bland-Altman analysis. 99.6% of the HbA1 levels for paired HemeChip-GHb and HPLC fell within A and B zones of no difference in clinical outcome based on error grid analysis. Using HemeChip-GHb we achieved accurate diabetes status detection with sensitivity and specificity of 100%. We presented a novel POC paper-based affinity electrophoresis platform that has the potential for accurately diagnosing diabetes, and addressing an unmet need for accurate and affordable diagnostics in resource-challenged environments.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate-binding modules (CBMs). In recent years carbohydrate surface binding sites of proteins, mostly enzymes, have also been investigated by this method. Here we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Carbohydrate-binding modules of ChiB and ChiC promote the chitinolytic system of Serratia marcescens BWL1001

Carbohydrate-binding modules (CBMs) are commonly found within chitinases, but their contributions to chitinolytic systems are poorly understood. To address this knowledge gap, full-length chitin-acting enzymes (ChiA, ChiB, ChiC, and CBP21) of Serratia marcescens BWL1001 and CBM-truncated versions (ChiB-dCBM and ChiC-dCBM) were heterologously expressed for enzymological analysis. The CBM5 of ChiB and the CBM12 of ChiC both exhibited an affinity for α-chitin, while only CBM12 could bind colloidal chitin based on adsorption assays and affinity electrophoresis. Consistent with their ligand specificity, both CBMs were essential to α-chitin hydrolysis, while only CBM12 enhanced the hydrolytic efficiency of colloidal chitin by individual chitinases. Analysis of synergistic hydrolysis with separate full-length and CBM-truncated chitinases revealed that the two CBMs promoted the synergistic activity of chitinases on crystalline and amorphous chitin. The two CBMs also promoted the hydrolysis when chitin was mixed with non-substrate polysaccharides. This study reveals not only the effects of CBMs on enzymatic characteristics of individual chitinases, but also their contributions to the overall efficiency of chitinolytic systems during synergistic hydrolysis.

Stream broadening in free flow affinity electrophoresis

Complexation plays a vital role in a variety of chemical and biological systems. Thus, our ability to reliably measure the equilibrium and kinetic parameters governing complexation reactions is crucial to comprehending important natural and artificial processes. Free-Flow Affinity Electrophoresis or FFAE offers a simple and powerful approach to measuring complexation parameters at high throughputs over a wide range of experimental conditions. However, the range and accuracy of analytical information that can be extracted from these affinity measurements remains limited due to significant knowledge gaps in our understanding of analyte transport in FFAE systems. To fill this gap in knowledge, the current article describes a mathematical formulation for quantitating stream broadening in FFAE assays. A closed-form expression has been derived for the stream width based on this formulation in the Taylor-Aris dispersion limit assuming fast analyte-ligand binding kinetics. It has been shown that the effects of affinity interactions on the stream dispersion in FFAE devices can be described using four terms that are additive to the contributions observed in conventional free-flow electrophoresis. Notably, all these additional terms were determined to scale with the square of the distance migrated by the analyte stream in the lateral direction. Moreover, they were shown to vary inversely with the Damköhler number in the system computed as the ratio of the rate of reaction over that of diffusive mass transfer.

Recent developments in capillary and microchip electroseparations of peptides (2019-mid 2021).

The review provides a comprehensive overview of developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, microscale isolation, and physicochemical characterization of peptides from 2019 up to approximately the middle of 2021. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis, such as sample preparation, sorption suppression, EOF control, and detection, are presented. New developments in the individual CE and CEC methods are demonstrated and several types of their applications are shown. They include qualitative and quantitative analysis, determination in complex biomatrices, monitoring of chemical and enzymatic reactions and physicochemical changes, amino acid, sequence, and chiral analyses, and peptide mapping of proteins. In addition, micropreparative separations and determination of significant physicochemical parameters of peptides by CE and CEC methods are described.

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

Role of Serine 263 in the Human Choline Transporter 1 and Congenital Myasthenic Syndrome

Disruptions of the neuromuscular junction (NMJ) result in congenital myasthenic syndromes (CMS). CMS is characterized by a range of phenotypes including muscle weakness, apnoeas, akinesia or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). CHT1 re-uptake of choline is a requirement for acetylcholine synthesis and NMJ function. The 13 transmembrane domain CHT1 protein is also thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive variant c.C788>T in SLC5A7 (NM_021815.4), in a family with a history of CMS using whole exome sequencing. This variant encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether a change in phosphorylation and/or charge/size of position 263 results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetics p.S263D and p.S263E. Upon expression in HEK293 cells and the utilization of cell surface biotinylation experiments, we found that WT, p.S263F and p.S263A mutants were present at the plasma membrane. Radioactive transport assays indicated that the p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non-functional. Immobilized metal affinity electrophoresis suggests that wild-type CHT1 may be more phosphorylated than the p.S263F CHT1 mutant. These results support the essential role of the Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status as well as size/charge at this position.

Binding modes of cibacron blue with albumin in affinity chromatography using docking tools

Affinity chromatography is a standard method, which used for protein purification and separation studies due to its specificity and selectivity. There are several affinity chromatography methods, such as dye affinity, immobilized metal chelated affinity, and affinity electrophoresis. Cibacron Blue F3G-A (CBD), as a dye ligand, is one of the most used dyes among dye affinity chromatography. CBD is ideally suited for human serum albumin (HSA) separation and purification in affinity chromatography for several years. However, even though CBD has many purification applications, there is not much research focused on the interaction between CBD and HSA in molecular docking. The interactions between CBD and HSA were simulated via AutoDock molecular docking software in this study. Investigated possibilities resulted in six different conformations on different locations, which light the way to variable connectivity of CBD. Thus, it was determined that the most favorable binding is conformation 5, with its lowest binding energy, which is energetically favorable.

Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter’s function

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788>T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status.Support or Funding InformationWomen and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research & The University of Alberta

Molecular insight into a new low-affinity xylan binding module from the xylanolytic gut symbiont Roseburiaintestinalis.

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of RiCBM86 appended to a GH family 10 xylanase from Roseburiaintestinalis. This CBM represents a new family of xylan binding CBMs present in xylanases from abundant and prevalent healthy human gut Clostridiales. RiCBM86 adopts a canonical β-sandwich fold, but shows structural divergence from known CBMs. The structure of RiCBM86 has been determined with a bound xylohexaose, which revealed an open and shallow binding site. RiCBM86 recognizes only a single xylosyl ring with direct hydrogen bonds. This mode of recognition is unprecedented amongst previously reported xylan binding type-B CBMs that display more extensive hydrogen-bonding patterns to their ligands or employ Ca2+ to mediate ligand-binding. The architecture of RiCBM86 is consistent with an atypically low binding affinity (KD about0.5mm for xylohexaose) compared to most xylan binding CBMs. Analyses using NMR spectroscopy corroborated the observations from the complex structure and the preference of RiCBM86 to arabinoxylan over glucuronoxylan, consistent with the largely negatively charged surface flanking the binding site. Mutational analysis and affinity electrophoresis established the importance of key binding residues, which are conserved in the family. This study provides novel insight into the structural features that shape low-affinity CBMs that mediate extended bacterial glycan capture in the human gut niche. DATABASES: Structural data are available in the protein data bank database under the accession number 6SGF. Sequence data are available in the GenBank database under the accession number EEV01588.1. The assignment of the Roseburiaintestinalis xylan binding module into the CBM86 new family is available in the CAZy database (http://www.cazy.org/CBM86.html).

Recent developments in capillary and microchip electroseparations of peptides (2017-mid 2019).

The review presents a comprehensive survey of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical and biochemical characterization of peptides since 2017 up to about the middle of 2019. Progress in the study of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression, EOF control, and detection) are described. Advances in CE and CEC methods are demonstrated and their applications in the following areas are presented: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. In addition, micropreparative separations and determinations of important physicochemical characteristics of peptides by CE and CEC methods are reported.

Heterologous expression of Thrombocidin-1 in Pichia pastoris: Evaluation of its antibacterial and antioxidant activity

The antimicrobial peptide Thrombocidin-1 (TC-1) isolated from human blood that derived from NAP-2 by deleting of two amino acids from C-terminal region. In this study, a C-terminal 6 _ His tagged recombinant TC-1 was expressed as a secreted peptide in Pichia pastoris, for the first time. The recombinant P. pastoris was inoculated in to BMMY culture medium, incubation with 5 μl/ml absolute methanol for 72 h at 30 °C. The TC-1 peptide was concentrated with nickel affinity chromatography and electrophoresis on 16% acrylamide gels. The molecular weight of recombinant TC-1 is approximately 8 kDa and under these conditions, the concentration of TC-1 considered 190 μg/ml that determined by the Bradford method. The antimicrobial activity test (Minimum Inhibitory Concentration and Minimum Bactericidal Concentration) was done against: Listeria monocytogenes, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The growth of these pathogenic bacteria was limited when we used peptide at a concentration of as low as 19.56 μg/ml. Based on DPPH radical scavenging (DPPH-RS) activity and reducing power assays, this peptide showed relatively good antioxidant potential in comparison with standard antioxidant used in this study (BHT). Due to the existence of TC-1 in blood, which makes it safe for human consumption, and the good results of its antimicrobial and antioxidant activity, it can be introduced as a good alternative and a novel effective peptide to food industry for bio-preservation.

Investigation of binding behavior of important metal ions to thioredoxin reductase using mobility-shift affinity capillary electrophoresis: A preliminary insight into the development of new metal-based anticancer drugs

Thioredoxin reductase (TrxR) is a redox enzyme that is found to be elevated in a variety of tumors and its inhibition has been proved to be a new approach for the treatment of cancer. In this study, the binding behaviors of some metal ions on TrxR have been evaluated using mobility-shift affinity electrophoresis. Eighteen medicinally important metal ions from soft, borderline and hard categories were selected for the screening. This study was based on the fact that, overall mass, charge and size of a protein changes after interaction with charged ligands such as metal ions, which affects the electrophoretic movement of the analyte protein under the influence of electroosmotic flow. This alteration in electrophoretic mobility of the analyte is proportional to the strength of interaction between the two species and is utilized to calculate the binding results. The mobility ratio (R) of an EOF marker (injected in same run) and the analyte enzyme was determined and the binding result was expressed as the normalized difference of the mobility ratios of test enzyme with and without metal ion (ΔR/Rf) and its confidence interval. The method showed excellent precision, as the % RSD of mobility ratios of replicate runs (n = 6) for most of the enzyme-metal ion pair was found to be <1%. Six out of eighteen tested metal ions including Ir3+, Ag+, Os3+, Cr3+, Fe2+ and Rh3+ showed significant binding interactions (ΔR/Rf ≥ ±0.01), while for others, it was found to be weak or insignificant. Among all the tested metal ions, Ir3+ (ΔR/Rf = −0.27065 ± 0.00818) showed the strongest interaction followed by Ag+ (ΔR/Rf = −0.09821 ± 0.00783), Fe2+ (ΔR/Rf = −0.0482 ± 0.00701), Os3+ (ΔR/Rf = −0.04382 ± 0.00751), Cr2+ (ΔR/Rf = −0.04093 ± 0.01206) and Rh3+ (ΔR/Rf = −0.01455 ± 0.00857). The values of ΔR/Rf obtained for most of the enzyme-metal ion pair were negative, suggesting the multifaceted complexation behavior of metal ions, as they get coordinated with surrounding anions after initial binding with the enzyme. As a result, the overall charge on the enzyme becomes more negative. Significant interactions of the enzyme with some metal ions suggested that the drugs based on these metal ions may prove to be potential inhibitor of the enzyme, however, it would be interesting to evaluate that how these interactions would affect the function and stability of the enzyme.

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.