Rapid Screening and Estimation of Binding Constants for InteractionsofFewithTwoMetalloproteins,Apotransferrinand Transferrin, Using Affinity Mode of Capillary Electrophoresis

*e interaction behavior of Fe with transferrin and apotransferrin (iron-free form) was investigated in this study using affinity capillary electrophoresis. Change in the mass and charge of protein upon binding to the metal ion in the capillary tube led to variation in its migration time and was used to measure the noncovalent binding interactions by fast screening method. Acetanilide was used as the electroosmotic flow (EOF) marker to avoid possible errors due to the change in EOF during the experiment. *e binding results were calculated from the mobility ratios of protein (Ri) and EOF marker (Rf) using the formula (Ri−Rf )/Rf or ∆R/Rf. For more comprehensive understanding, the kinetics of the interaction was studied and binding constants were calculated. Results showed that the Fe displayed insignificant interaction with both proteins at lower metal ion concentrations (5–25 μmol/mL). However, transferrin exhibited significant interactions with the metal ion at 50 and 100 μmol/mL (ΔR/Rf� 0.0114 and 0.0201, resp.) concentrations and apotransferrin showed strong binding interactions (ΔR/Rf� −0.0254 and 0.0205, resp.) at relatively higher Fe concentrations of 100 and 250 μmol/mL. *e binding constants of 18.968mmol and −13.603mmol were recorded for Fe interaction with transferrin and apotransferrin, respectively, showing significant interactions. Different binding patterns of Fe with both proteins might be attributed to the fact that the iron-binding sites in transferrin have already been occupied, which was not the case in apotransferrin. *e present study may be used as a reference for the investigation of protein-metal ion, drug-protein, drug-metal ion, and enzyme-metal ion interactions and may be helpful to provide preliminary insight into the new metal-based drug development.


Introduction
Iron is essential for biological systems as it plays an important role as a cofactor in several important biological processes, including respiration and DNA replication. It is insoluble and toxic in the free iron (III) ion form. Transferrin is an iron-transport protein that is crucial for the delivery of iron (Fe 3+ ) to the cells. In the process of iron transportation, iron-bound transferrin (holofrom) interacts with transferrin receptors at the cell surface and gets transported to endosomes, where iron is released due to acidic pH. Iron-free transferrin (apofrom) gets back to the cell surface and dissociates from the receptor [1][2][3]. Iron (III) ion preferably binds to the hard functional groups, including aspartate, glutamate, tyrosine, and histidine amino acid residues [4]. Conversely, the iron ion of low oxidation state (Fe 2+ ) favorably interacts with moieties containing borderline acid groups such as porphyrin residue in hemoglobin. e hard and soft acid and base (HSAB) principle can explain the mechanism of metal ionligand complexation reactions in a qualitative manner and provide information regarding the stability of their complexes [5]. e classification of metal ions and other ligands into hard and soft acid-base along with their characteristics and binding preferences according to the HSAB principle is represented in Figure 1. Transferrin, an iron-transport protein, consists of iron-binding nonheme glycoproteins widely distributed in the physiological fluids and cells of organisms. e main function of transferrin is to transport Fe 3+ through the circulatory system and deliver it to cells by endocytosis [6,7]. It consists of two similar but nonidentical metal ion-binding locations present in N-and C-terminal domains. Generally, transferrin interacts with two Fe 3+ ions to produce a pinkcolored complex in the presence of bicarbonate (or carbonate) ions. e binding of Fe 3+ ion to transferrin causes a significant conformational alteration in the protein structure, resulting in the conversion of the open (apoprotein) form to the closed (holoprotein) conformation. Apotransferrin is known as an iron-depleted form of transferrin. Only the holoform of transferrin can bind to the receptor, while it could not recognize the apotransferrin. Transferrin can also interact with a number of di-, tri-, and tetravalent metal ions, including those from transition, lanthanide, and actinide series, with a mechanism similar to that of Fe 3+ -transferrin interaction. Evidence indicating the transferrin cycle as an important cellular uptake pathway for several clinically important metal ions is available. Furthermore, transferrin is often viewed as promising delivery means for cytotoxic metal ions and metal ion-based therapeutics to malignant cells [8][9][10].

Analytical Techniques for Protein-Metal Interaction Study.
Proteins are generally complex in nature, rendering the investigation of protein-metal ion interaction a challenging task. Various analytical techniques, including NMR spectroscopy, FTIR spectroscopy, X-ray crystallography, atomic force microscopy (AFM), circular dichroism (CD) spectroscopy, and surface plasmon resonance (SPR), have been used for the investigation of protein-metal ion interactions [6,7]. e interaction of proteins with molecular weights of <40 kDa can be easily analyzed with the NMR technique. However, for larger proteins (molecular weight ≥40 kDa), isotopically labeled samples are used, which make the analysis complicated [11], whereas considerably longer time (4-5 weeks) is needed in the sample preparation in X-ray crystallography; even in some cases, the crystallization is very difficult [12]. Both X-ray crystallography and NMR spectroscopy are more preferred for structure elucidation and may be used further to verify the results obtained from other techniques. Affinity capillary electrophoresis (ACE) and affinity chromatography are two important techniques used recently for studying protein-metal ion bindings. Because of their good separation capabilities, these techniques are advantageous when protein samples are impure, including a biological sample. However, with affinity chromatography, cost considerations become important owing to the use of affinity columns besides a large amount of sample for analysis [13,14]. Recently, ACE was successfully used for studying the interaction of various proteins with different metal ions [15][16][17][18]. Various isoforms of proteins were also investigated simultaneously using ACE, which   Journal of Spectroscopy could easily separate each isoform in relatively lesser separation times [19]. ACE-UV has been considered to be a good option for studying protein-metal ion interactions due to easy handling, cost-effectiveness, and reliable results. Additional advantages of ACE include the requirement of a very small volume of sample, high separation efficiency, and the ability to perform the experiment at pH 7.4 and 37°C, closely depicting the native environment of proteins under physiological conditions. Due to excellent separation efficiency, proteins and other biological samples can be analyzed directly in their impure form, minimizing the sample preparation time [20][21][22][23][24].

Affinity Capillary Electrophoresis (ACE)
. ACE is used to study the relative affinities of two reactants using the separation power of the capillary electrophoresis. Recently, ACE gained much attention in studying protein-metal ion interactions and determining their binding constants [25][26][27]. It is generally utilized to study noncovalent binding interactions between an analyte and a ligand, where the alteration in electrophoretic migration (mobility shift), area, and/or height of the analyte peak before and after binding is measured. One of the three ACE modes, namely, preequilibrated, dynamic equilibrium, and kinetic modes, may be used to investigate the interaction behavior of protein and metal ions. Dynamic equilibrium mode is generally preferred when the relaxation time of metal ion binding is shorter than electrophoretic separation time. e interaction between analyte and ligand takes place in the capillary tube during the electrophoretic run; hence, the dynamic mode has the advantage of being faster than other modes (no extra time for interaction is required). Dynamic equilibrium mode using mobility shift ACE was selected for this study because it is highly sensitive and is capable of detecting even very weak interactions correctly. e precision of the method can further be increased by calculating the mobility ratio for the interaction screening [15][16][17][18][19]. In ACE, the mobility ratio (R), which is the ratio of the analyte mobility (μ) and can be expressed as where R is the mobility ratio and μ prot and μ eof represent the mobility of protein and EOF marker, respectively. e mobility can be calculated by where "V" represents the applied voltage and "x" and "D" are the effective and total length of the capillary tube, respectively. All these factors except migration time "t" are common for both protein and EOF marker and hence will be canceled. us, the mobility ratio of the interaction can be calculated using the recorded migration time by where t eof and t prot represent the migration time for EOF marker and protein, respectively [19,22]. Although the interaction of Fe 3+ ion with transferrin has been investigated using various analytical techniques [28][29][30], no study based on ACE has been conducted so far. erefore, the present work was commenced to study the interaction behavior of Fe 3+ with transferrin and apotransferrin using fast screening method by mobility shift affinity capillary electrophoresis and determine the binding constants for better understanding of interaction behavior. e current study will provide a reference for the application of ACE in the investigation of different types of interactions such as enzyme-metal ion, drug-protein, drug-enzyme, and protein-protein interactions, in addition to determining the binding behaviors of biologically significant metal ions to various metalloproteins. Consequently, this study may prove to be useful to afford preliminary information in the development of new metal-based therapeutic agents.

Instrumentation.
Agilent Capillary Electrophoresis (CE) system, model no. G1600A (Agilent Technologies, Germany), was used to screen protein-metal ion interactions by recording electrophoretic mobility of the analyte protein before and after complexation. e CE instrument was equipped with a capillary cooling system, an autosampler, and a diode-array detector which was operated at 214 nm wavelength. Electrophoretic migration was performed on a bare-fused silica capillary tube with an effective and total length of 22 cm and 31 cm, respectively (short capillary), and an internal diameter of 50 μm. e capillary was procured from Polymicro Technologies (Phoenix, AZ, USA). A normal air plug was utilized to apply high pressure (2.5 bars) at the laboratory. e pH of the solutions was adjusted using the Jenway pH meter (Cole-Parmer, Staffordshire, United Kingdom). ROTILABO CME syringe filters (25 mm dimension and 0.22 μm pore size) were procured from Carl Roth, Germany. e instrument was monitored, screening data was processed by ChemStation software (Agilent Technologies, Germany), and Microsoft Excel version 2010 (Microsoft Corporation, USA) was used for simulation of different interaction parameters, including mobility ratios (R), ∆R/Rf, and confidence interval.

Rinsing Procedure for Regeneration of Capillary.
Initially, the new capillary tube was conditioned for 20 min at 1 bar with 1 N sodium hydroxide solution followed by ultrapure water. At the beginning and end of each working day, extra flushing with 0.1 N NaOH solution for 10 min followed by water for 5 min at 2.5 bar was performed. Furthermore, at the beginning of each analysis, capillaries were conditioned with a mixture of 0.1 M EDTA and 0.1 N NaOH solutions at 2.5 bar for 2.5 min, ultrapure water for 1.0 min, and running buffer for 1.5 min.

Electrophoretic
Conditions. Electrophoretic migration of analyte was achieved by applying a voltage of 10 kV at 23°C capillary temperature. e separation was performed at electrophoretic mode, where anode was at the inlet and cathode at the outlet of the capillary (normal mode). Sample solutions were injected into the capillary at 50 mbar using the hydrodynamic mode, and after sample injection, the running buffer was injected at 50 mbar for 2.5 s. Overall, twelve runs were completed for each sample, including six runs for analyte protein with Fe 3+ and six runs without metal ion.

Preparation of Analytical Solution
2.5.1. Tris Buffer (pH 7.4, 20 mmol/L). Accurately weighed 2.42 g of tris powder was dissolved in approximately 200 mL double distilled water. e pH of the solution was adjusted with dilute HCl to 7.4, and the volume was adjusted to 1000 mL using water.

Acetanilide Stock Solution.
Accurately weighed 37.5 mg of acetanilide was transferred in 50 mL tris buffer (pH 7.4) and dissolved completely by sonication to obtain a concentration of 750 μg/mL.

Protein Working Solutions.
Appropriate quantities of transferrin and apotransferrin were taken separately into 25 mL volumetric flasks and dissolved in small amounts of tris buffer. Aliquots of 5 mL of acetanilide stock solution were added to each flask, and the volumes were completed with tris buffer to obtain solutions having 15 μmol/L concentrations for both proteins. To minimize protein adsorption on the wall of the capillary and band broadening, the above protein solutions were diluted threefolds in tris buffer.

Iron (III) Ion
Solution. An appropriate amount of ferric chloride was dissolved in a solution containing 20 mmol/L tris buffer (unadjusted pH) and 15 mmol/L malonic acids to achieve a stock solution of 5 mmol/L concentration of metal ion (Fe 3+ ). As iron (III) is not properly soluble in tris buffer and tends to precipitate; hence, malonic acid was added to assist the dissolution of ferric chloride. Malonic acid weakly chelates the iron (III) ions leading to the formation of a complex which could exchange the ligand metal ions with protein residues [19]. e metal ion solution was then diluted in tris buffer (pH 7.4, 20 mmol/ L) to obtain working solutions of 5, 10, 25, 50, 100, and 250 μmol/L concentrations. e protein and metal ion solutions could be used for only one day; consequently, all the solutions were freshly prepared at the beginning of each working day. All the prepared solutions were filtered using 0.22 μm syringe filter before analysis.

Fast Screening by Normalized Difference of Mobility
Ratios. In ACE, migration time (mobility) of analyte protein is changed because of the variation in overall mass and charge following complexation with metal ions.
is has been used as a measure of the extent of interaction between the metal ions and test proteins. Protein adsorption on the inner surface of the capillary tube often reduces the precision of binding results. Furthermore, binding of protein analyte with the inner capillary surface may lead to alteration in the electrophoretic mobility during electrophoretic run [31,32]. erefore, to minimize possible errors in the results caused by variation in the electrophoretic mobility of the protein, an EOF marker is generally used. In this work, acetanilide, which is neutral at physiological pH (7.4), was used as an EOF marker, and mobility ratios of proteins and EOF marker (μ prot /μ eof ) were used to measure the interaction. In order to reduce protein adsorption and to obtain good interaction results, an appropriate rinsing procedure as mentioned above in the rinsing protocol section was implemented.
In the current study, the interaction between Fe 3+ ion and two transport proteins, transferrin and apotransferrin, was screened, and the interaction results were then evaluated by their mobility ratios with precise results. e presence of consistent noncovalent interactions was confirmed by a change in the electrophoretic migration of proteins after interaction with Fe 3+ ion ( Figure 2). e strength of interactions was calculated as the difference of mobility ratios of protein before and after interaction with a metal ion (Rf and Ri, resp.) normalized by Rf (∆R/Rf; ∆R � Rf − Ri) and confidence intervals (cnf ), by the calculation method described by Alhazmi et al. [19]. Even a small change in protein mobility is enough to detect an interaction with a ligand metal ion, and a calculated value of ΔR/Rf ≥ 0.01 either in positive or negative direction proves a significant interaction [19].
Transferrin and apotransferrin interacted with iron (III) ion and electropherograms thus obtained were recorded. Metal ion concentrations of 5, 10, 25, 50, 100, 150, and 250 μmol/L were used for the study, and the results are summarized in Tables 1 and 2. At each metal ion concentration, 12 runs were performed for both protein samples (six without metal ion and six with metal ion), and corresponding mobility ratios (Rf and Ri) were calculated. It is evident from Table 1 that the % relative standard deviations (%RSDs) of the interaction results were less than 2% for both test proteins at most of the metal ion concentrations, demonstrating high precision results. Among the investigated metal ion concentrations, significant binding interactions (ΔR/Rf ≥ 0.01) were observed at 50 and 100 μmol/L with transferrin, while at concentrations of 100 and 250 mmol/L, apotransferrin showed the highest interactions. At other concentrations, the metal ion showed weaker affinities (ΔR/Rf < 0.01) towards both test proteins; however, significantly smaller confidence intervals were observed at all the tested concentrations of the metal ion. e negative and positive signs of ∆R/Rf values as recorded in the screening results are also important as they could give valuable information about the coordination of bound metal ions with other protein residues and/or surrounding anions in the capillary tube. In general, an absolute value of ΔR/Rf ≥0.01 is enough to detect remarkable interactions [19,33]. More importantly, for a significant protein-metal ion interaction, the value of cnf of ΔR/Rf should not overpass the zero values in ΔR/Rf ± cnf. In normal circumstances, a positive value of ΔR/Rf is achieved because the overall charge on the protein will get more positive (less negative) after binding to the metal ion. However, a negative value of ΔR/Rf could alternatively be obtained when the bound metal ions on the   Journal of Spectroscopy protein further interact coordinatively with the surrounding anions present in the medium, which may result in a less positive (more negative) overall charge on the protein molecule. As shown in Table 2, some interesting results were obtained for the interaction of test proteins with iron (III) ions. At lower concentrations (5, 10, and 25 μmol/L) of metal ion, insignificant interaction with transferrin (ΔR/ Rf � −0.007, 0.0012, and −0.007, resp.) and apotransferrin (ΔR/Rf � −0.0024, 0.0021, and −0.0008, resp.) was observed. A significant interaction between iron (III) ion and transferrin was recorded at 50 and 100 μmol/L of metal ion concentrations (ΔR/Rf � 0.0114 and 0.0201, resp.), whereas the test metal ion exhibited significant binding affinities with apotransferrin at 100 and 250 μmol/L concentrations (ΔR/ Rf � −0.0254 and 0.0205, resp.). Interestingly, both proteins showed the strongest binding interactions at 100 μmol/L iron (III) ion concentration. For most of the interactions, negative ΔR/Rf values were observed, indicating that the protein-bound metal ions further coordinate with the adjacent anions present in the medium rather than other residues at the binding site of the protein and therefore the overall charge on the proteins get more negative. Furthermore, metal ion interaction with other high-affinity amino acid residues at the binding site is also possible. Proteinmetal ion complexes are formed by interaction at more than one amino acid residue, and the binding sites for metal ions can be confirmed using NMR spectroscopy and X-ray crystallographic techniques [34]. At 50 μmol/L metal ion concentration, the overall charge on both proteins becomes more positive after interacting with the Fe 3+ ion, and therefore, positive values of ΔR/Rf were obtained. is can be explained by the fact that the number of bound metal ions coordinating with binding site residues is higher than those with the surrounding anions. e variation of results from negative to positive ΔR/Rf values and vice versa is might be due to the availability of multiple metal ion-binding sites on proteins with different binding properties. In addition to alteration in the migration time, slight variation in size and shape of the peaks due to protein-metal ion complex was also recorded at certain metal ion concentrations. is may be due to the conformational changes in protein structure as a result of interaction with metal ion (Fe 3+ ). Change in peak shape may also be attributed to variation in pH or ionic strength of the medium [31,32].

Kinetics Binding Constants.
Upon confirmation of significant noncovalent interactions between Fe 3+ and test proteins, binding constants of the interactions were estimated. Four mathematically plotted models are used to estimate the binding constants of ligand-biomolecule interaction, which are nonlinear regression, X-reciprocal, Y-reciprocal, and double-reciprocal methods as shown in equations (4)-(7) [22,35]: (a) Nonlinear regression: (b) X-reciprocal: (d) Double-reciprocal: where K is the binding constant; c (L) is the micromolar concentration of ligand (metal ion); Rf and Ri are mobility ratios of the protein before and after interacting with the ligand (metal ion) in the capillary tube, respectively; Rc represents the mobility ratio of analyte protein at saturated ligand concentration. All these plotting methods have different statistical treatment of data points as depicted in Table 3. Since a ligand especially metal ion can bind to multiple target sites on the proteins, 1 : 1 binding stoichiometry is uncommon. erefore, in the screening of binding interaction between protein and metal ions using the ACE technique, nonlinear regression analysis was found to be the most appropriate method. Moreover, in ACE analysis, estimation of association and dissociation constants is less sensitive to random error while using the nonlinear regression method. e literature also revealed that the cumbersome weighted regression procedure is avoided in the nonlinear regression method, which is necessary for the statistical analysis of the other three methods (linearized plots) [36]. Consequently, in the present investigation, the nonlinear regression method was used for the estimation of binding constants. Metal ion concentrations in the range of 5-250 μmol/L were used to calculate the binding constants. As shown in Table 3, the binding constant can be calculated using Rc,  which is the mobility ratio of saturated protein measured at the highest metal ion concentration. A Fe 3+ ion concentration of 250 μmol/L was used for both transferrin and apotransferrin. e mobility ratios obtained at 250 μmol/L Fe 3+ concentration were used in the nonlinear regression analysis, and the binding constants were calculated to be 18.968 mmol −1 and −13.6031 mmol −1 for transferrin and apotransferrin, respectively. e binding plots were drawn between (Rf − Ri)/(Ri − Rc) and ligand (metal ion) concentration [22,35], and the progress of interaction was then analyzed using respective binding curves. Interestingly, the Fe 3+ ion was observed to behave differently with the two test proteins (Figure 3). As discussed in the case of ΔR/Rf, values, the positive and negative signs of the binding constant are attributed to the behavior of metal ions at different binding sites in the tested proteins and the additional coordination with surrounding background ions. e negative and positive values of binding constants indicate the direction of the binding interaction between the metal ion and protein. e negative sign does not mean that the value is less than zero and be considered as noninteraction. Hence, the binding constant value can be used for the characterization of protein-metal ion interaction [22]. e nonlinear regression curve revealed that the transferrin exhibited insignificant binding interaction with Fe 3+ at initial concentrations (5, 10, and 25 μmol/L), whereas linearly increasing affinity was observed at metal ion concentrations of 50 and 100 μmol/L and a positive value of binding constant (18.968 mmol −1 ) was obtained. On the other hand, a negative value of the binding constant (−13.603 mmol −1 ) was obtained for the interaction between Fe 3+ and apotransferrin. At the initial metal ion concentration (5 μmol/L), a positive and slightly stronger binding than transferrin was observed in case of apotransferrin, while it changed to be negative at 10 μmol/L and again positive at 25 μmol/L. Finally, the binding affinity between the metal ion and apotransferrin sharply increased at 100 and 250 μmol/L metal ion concentrations, and the binding pattern was shifted to a less negative direction. Different signs of the binding constants, positive for transferrin and negative for apotransferrin, and nonidentical patterns of binding curves could be attributed to different target binding sites on the tested proteins. e tested metal ion binds to nonspecific binding sites on transferrin (specific binding sites already occupied by Fe 3+ ), whereas both specific and nonspecific binding sites in case of apotransferrin lead to different interaction behaviors of Fe 3+ on both tested proteins.
Overall, the use of fast screening by the normalized difference of mobility ratios is inadequate for studying and comparing the binding of metal ions to proteins. Consequently, in the present study, the fast screening method showed unclear binding results between the tested proteins and Fe 3+ . An insignificant binding interaction was observed at lower iron (III) ion concentration with both tested proteins. Transferrin exhibited greater affinities at medium concentrations (50 and 100 μmol/L), whereas apotransferrin was found to show stronger interactions at higher metal ion concentrations (100 and 250 μmol/L). e interaction behavior became more evident from the kinetic study of the interaction. e calculation of binding constants and curve patterns for the interaction between proteins and iron (III) ion revealed that the metal ion exhibited significant binding affinities with both proteins; however, it behaved differently due to differences in the availability of the specific binding sites on both proteins.

Conclusion
In the present study, noncovalent interactions between two important metal transport proteins and iron (III) ions were investigated using the ACE technique. During the analysis, inner capillary wall was intermittently regenerated by applying proper rinsing protocol, the low voltage was applied to decrease joule heat, and use of a short capillary tube has offered a fast and precise analysis. e fast screening method was based on the calculation of the normalized difference of mobility ratios (∆R/Rf ) of protein and electroosmotic flow marker, representing the binding results (a value of ∆R/ Rf ≥ 0.01 indicates significant binding). Transferrin showed significant binding interaction at 50 and 100 μmol/L concentrations of iron (III) ion, whereas apotransferrin exhibited remarkable binding affinity to metal ion at 100 and 250 μmol/L concentrations. Both proteins showed insignificant affinities at lower metal ion concentrations (5-25 μmol/L). In addition to that, binding constants of the interaction of iron (III) ion and test protein were estimated to obtain a more comprehensive understanding of the affinity of metal ions to the proteins. e binding constants of 18.968 mmol −1 and −13.603 mmol −1 /L for iron (III) ion interaction with transferrin and apotransferrin, respectively, were obtained. e positive and negative values of binding constants indicated different behaviors of Fe 3+ ion with transferrin and apotransferrin, which may be attributed to the difference in the availability of specific and nonspecific binding sites on both proteins. e outcomes of the present investigation recommended the use of kinetic study in combination with the fast screening method in the ACE technique to understand the interactions between proteins and metal ions.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.