FT-IR Spectroscopy for the Identification of Binding Sites And

Scientia Pharmaceutica

Article FT-IR Spectroscopy for the Identiﬁcation of Binding Sites and Measurements of the Binding Interactions of Important Metal Ions with Bovine Serum Albumin

Hassan A. Alhazmi

Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; [email protected]; Tel.: +966-541433344

Received: 15 January 2019; Accepted: 13 February 2019; Published: 20 February 2019

Abstract: Proteins play crucial roles in the transportation and distribution of therapeutic substances, including metal ions in living systems. Some metal ions can strongly associate, while others show low affinity towards proteins. Consequently, in the present work, the binding behaviors of Ca2+, Ba2+, Ag+, Ru3+, Cu2+ and Co2+ with bovine serum albumin (BSA) were screened. BSA and the metal ions were allowed to interact at physiological pH and their binding interactions were screened by using FT-IR spectroscopy. Spectra were collected by using hydrated films over a range of 4000–400 cm−1. The interaction was demonstrated by a significant reduction in the spectral intensities of the amide I (C=O stretching) and amide II bands (C–N stretching coupled to NH bending) of the protein after complexation with metal ions. The binding interaction was further revealed by spectral shifting of the amide I band from 1651 cm−1 (free BSA) to 1653, 1654, 1649, 1655, 1655, and 1654 cm−1 for BSA–Ca2+, BSA–Ba2+, BSA–Ag+, BSA–Ru3+, BSA–Cu2+ and BSA–Co2+ complexes, respectively. The shifting of the amide I band was due to the interactions of metal ions with the O and N atoms of the ligand protein. Estimation of the secondary protein structure showed alteration in the protein conformation, characterized by a marked decrease (12.9–40.3%) in the α-helix accompanied by increased β-sheet and β-turn after interaction with the metal ions. The interaction results of this study were comparable with those reported in our previous investigation of metal ion–BSA interactions using affinity capillary electrophoresis (ACE), which has proven the accuracy of the FT-IR technique in the measurement of interactions between proteins and metal ions.

Keywords: bovine serum albumin; FT-IR; metal ions; secondary structure; binding interaction

1. Introduction Metal ions play crucial roles in maintaining physiological functions inside the human body and are used for diagnostic as well as therapeutic purposes. Some metals are essential for life and their deficiency in the biological system may lead to a variety of ailments. For instance, insufficient zinc in the diet leads to growth retardation and copper deficiency results in cardiovascular complications [1]. Considering their detoxifying capability, certain metal ions are utilized as chelating agents for the treatment of different types of diseases. Recently, metal-based complexes have been extensively investigated mainly for the development of anticancer agents and many of them have shown encouraging results. Cisplatin, a platinum (IV)-based drug, is the best example of such metal ion complexes and is currently in clinical applications for the treatment of various types of cancers [2]. The association of metal ions with proteins serves several important functions in the body, such as the transportation of biologically important metal ions to their specific target binding sites as one of the most crucial tasks. In a protein, the amino acid residues provide negatively charged functional groups as potential ligands for cationic metal ions. As a consequence, there exists an attraction between the

Sci. Pharm. 2019, 87, 5; doi:10.3390/scipharm87010005 www.mdpi.com/journal/scipharm Sci. Pharm. 2019, 87, 5 2 of 13 Sci. Pharm. 2019, 87, x FOR PEER REVIEW 2 of 13 betweenelectron-rich the electron functional-rich groups functional in proteins groups and in proteins electron-deficient and electron (cationic)-deficient metal (cationic ions,) leadingmetal ions, to a leadingbinding to interaction a binding betweeninteraction the between two species. the two Furthermore, species. Furthermore, the interaction the interaction of metal ions of tometal suitable ions tobiological suitable targetbiological binding target sites bindi alsong depends sites also on depends the valency on the and valency charge-accepting and charge capacity-accepting of thecapacity metal ofions the [3 metal]. Metal ions ion [3] interactions. Metal ion with interactions proteins maywith influenceproteins may the protein influence structure the protein and affect structure structural and affectstability. structural The impact stability. of metalThe impact ions binding of metal to ions proteins binding can to beproteins seen in can the be secondary, seen in the tertiary secondary, and tertiaryquaternary and structuresquaternary of structures the proteins. of the Albumin proteins. is theAlbumin major is plasma the major protein plasma in vertebrates, protein in vertebrates, comprising comprisingapproximately approximately 60% of the total60% of plasma the total content, plasma and content participates, and participate in the transportations in the transportation of a diverse of arange diverse of molecules, range of mo includinglecules, nutrients,including metal nutrients, ions, drugsmetal andions, metabolites. drugs and Duemetabolites. to their extraordinaryDue to their extraordinarybinding ability tobinding a variety ability of ligands, to a albuminsvariety have of ligands, several biochemical, albumins have pharmaceutical several biochemical, and clinical pharmaceuticalapplications [4, 5 and]. Bovine clinical serum applications albumin (BSA)[4,5]. isBovine among serum the most albumin commonly (BSA) investigated is among the proteins most commonlyfor research investigated purposes due proteins to its close for structural research purposes resemblance due to to human its close serum structural albumin resemblance (HSA). to humanStructurally, serum albumin the BSA (HSA). molecule has a single chain consisting of 583 amino acids, which is cross-linkedStructurally, through the 17BSA cystine molecule residues, has a and single its molecularchain consisting mass is of about 583 amino 66.5 kDa. acids, The which BSA moleculeis cross- linkedis made through up of three 17 cystine structurally residues identical, and its domains molecular (I, II mass and III), is about which 66.5 are dividedkDa. The into BSA nine molecule loops (L1 is madeto L9) up through of three disulfide structurally bonds. identical Every domain domains consists (I, II and of twoIII), subdomainswhich are divided (A and into B) and nine the loops loops (L1 in toeach L9) domain through are disulfide composed bonds. of a Every sequence domain of large, consists small of andtwo largesubdomains loops making (A and a B) triplet. and the The loops BSA inmolecule each domain predominantly are composed consists of a ofsequence a helical of structure, large, small with and about large 74% loops helical making content. a triplet. It also The hasBSA a moleculefree thiol grouppredominantly attached toconsists the Cys34 of a residuehelical structure [6,7]. The, with three-dimensional about 74% helical structure content. of BSA It also is shown has a freein Figure thiol 1group. attached to the Cys34 residue [6,7]. The three-dimensional structure of BSA is shown in Figure 1.

FigureFigure 1. 1. ThreeThree-dimensional-dimensional structure structure of of b bovineovine serum serum albumin albumin ( (BSA)BSA) [Source: [Source: protein protein data data bank; bank; ID: ID: 3V03V033 ( www.rcsb.org)www.rcsb.org)].].

TheThe sselectivityelectivity of of the interactions between between metal metal ions ions and and protein protein binding sites sites can can be best predicted as per the hard and soft acid base (HSAB) Lewis theory (Table (Table 11).). AccordingAccording toto thethe HSABHSAB LewisLewis theory, theory, metal metal ion ion ligands ligands bind bind preferentially preferentially to to the the protein protein binding binding sites sites of ofsimilar similar hardness hardness or softness.or softness. The The soft softmetal metal ligands ligands react react with with soft groups soft groups and hard and hardmetal metal ligands ligands interact interact with the with hard the bindinghard binding sites sitesof proteins of proteins [8,9] [.8 Generally,,9]. Generally, the theattraction attraction forces forces between between hard hard acid acid metals metals and and hard hard basicbasic protein sitessites areare ionic, ionic, whereas whereas covalent covalent interaction interaction exists exists between between soft soft acid acid metals metals and softand basicsoft basicprotein protein sites [sites8,10 ,11[8,10,11]]. The. interactionsThe interactions of metal of metal complexes complexes with with BSA BSA lead lead to the to disruptionthe disruption of the of thedisulﬁde disulfide bonds, bonds, altering altering the the secondary secondary structure structure of of proteins proteins and and result result inin unfoldingunfolding by significant signiﬁcant lossloss of of the the αα-helix-helix conformation conformation [12] [12. ].The The metal metal complexes complexes–protein–protein interactions interactions may may also also modify modify the overallthe overall polarity polarity of the of theenvironment environment around around the the exposure exposure of of tryptophan tryptophan residues, residues, which which is is due due to molecularmolecular interactions interactions such as as molecular molecular re rearrangements,arrangements, excited excited state state reactions, reactions, energy energy transfer transfer and and collisioncollision quenching [13, [13,14]14]..

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Table 1. Metal ions’ affinity to different donor ligands as per the hard and soft acid base (HSAB) Lewis theory. Sci. Pharm. 2019, 87, x FOR PEERTable REVIEW 1. Metal ions’ afﬁnity to different donor ligands as per the hard and soft acid base (HSAB) Lewis theory. 3 of 13 Hard Borderline Soft Hard Borderline Soft Acids Na+, K+, MgTable2+, Ca 1.2+ ,Metal Ba2+, Cr ions3+, Al’ affinity3+, Ga3+, Co to3+ different, Fe3+ donor ligandsCu2+, Zn as2+, Pbper2+ ,the Bi3+ ,hard Ni2+, andCo2+ ,soft Fe2+ acid base (HSAB)Cu+ ,Lewis Au+, Ag theory+, Hg2+., Hg+, Cd2+, Ru3+ Na+,K+, Mg2+, Ca2+, Ba2+, Cr3+, Al3+, Ga3+, Co3+, Fe3+ Cu2+, Zn2+, Pb2+, Bi3+, Ni2+, Co2+, Fe2+ Cu+, Au+, Ag+, Hg2+, Hg+, Cd2+, Ru3+ Acids High oxidation state Medium oxidation state Low oxidation state HighHard oxidation state MediumBorderline oxidation state Low oxidationSoft state AcidsBases Na-+COO, K+, -Mg, OH2+,- ,Ca -C=O,2+, Ba -2OH,+, Cr 3+-NH, Al23+, ,SO Ga4-3+2,, POCo3+4-3, ,Fe C–3+O -C, HCO3-1, Cu2+, ZnN 2+, Pb2+, Bi3+, Ni2+, Co2+, Fe2+ Cu+, Au+, Ag+, Hg2+, Hg+, Cd2+, Ru3+ HighH2O o xidationetc state Medium oxidation state Low oxidation state N H , -CONH2, Nitrogen of the peptide bond, -SH, R-S-R', , Bases -COO-, OH-, -C=O, -OH, -NH2, SO4-2, PO4-3, C–O-C, HCO3-1, N

H2O etc - - −2 −3 N N R C N -COO , OH , -C=O, -OH, -NH2, SO4 , PO4 , C–O-C, H , -CH=CH2 Bases −1 , -CONHH , NO2, 3Nitrogen-1, of the peptide bond, -SH, R-S-R', , HCO3 ,H2O etc

N N N R C N N NH2 , -CH=CH2 H , NO3-1, N N N N , NH2 N

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A number of analytical techniques, including X-ray crystallography, FT-IR spectroscopy, NMR spectroscopy, circular dichroism (CD) spectroscopy, UV–Vis absorption spectroscopy, fluorescence spectroscopy, ESI-MS, affinity chromatography and capillary electrophoresis, have been employed to investigate the binding interactions between metal ions and proteins [10,11,15,16]. FT-IR spectroscopy has been accepted as a powerful tool for the analysis of biomolecules. Due to the presence of characteristic functional groups, biomolecules such as proteins, carbohydrates, lipids and nucleic acids possess vibrational fingerprints at specific frequencies of infrared light. Hence, the structure and composition of these groups can be estimated by evaluating the intensity, position and width of the spectral bands in FT-IR [17–19]. To the extent of our knowledge, there is no report available regarding the analysis of the interaction between the selected biologically important metal ions and BSA. Hence, in the present work, we intended to study the interactions of group A metal ions (Ca2+ and Ba2+), noble metal ions (Ag+ and Ru3+) and heavy metal ions (Cu2+ and Co2+) with BSA using FT-IR spectroscopy. The change in the secondary structure of BSA was measured, which served as an effective tool to evaluate the qualitative as well as quantitative interactions between metal ions and BSA.

2. Materials and Methods

2.1. Chemicals and Instruments Bovine serum albumin (BSA, 99.0%) was procured from Sigma-Aldrich (Steinheim, North Rhine-Westphalia, Germany). Analytical grade tris powder, acetic acid, calcium (II) chloride (CaCl2), barium (II) nitrate {Ba(NO3)2}, silver (I) chloride (AgCl), ruthenium (III) chloride (RuCl3), copper (II) chloride (CuCl2), and cobalt (II) chloride (CoCl2.6H2O) were also purchased from Sigma-Aldrich (Steinheim, North Rhine-Westphalia, Germany) and used without further puriﬁcation. Ultrapure water (resistivity 18.2 Ω) was produced in-house using the milli-Q system (Millipore, Molsheim, France). The IR spectral analysis was carried out by using a FT-IR spectrophotometer (Nicolet iS10 FT-IR, Thermo Fischer Scientiﬁc, Waltham, Massachusetts, USA), equipped with a MCT liquid nitrogen cooled detector.

2.2. Preparation of Solutions Tris buffer (20 mM, pH 7.4) was prepared by dissolving 1.21 g of tris powder in 500 mL of ultrapure water. The pH 7.4 was maintained with dilute acetic acid. Then, 0.5 mM protein (BSA) solution was prepared by accurately weighing and dissolving BSA in 20 mM tris buffer (pH 7.4). Metal ion solutions of 1.0 mM concentrations were prepared in the above tris buffer and diluted to obtain solutions of 0.25, 0.1 and 0.025 mM concentrations. Metal ion solutions and protein solutions were prepared freshly every day of the experiment.

2.3. FT-IR Spectroscopic Analysis Metal ions and the protein (BSA) solution were mixed together by slowly adding the metal ion solutions to the BSA solution with continuous stirring. The solutions were mixed in such a way to achieve 0.125, 0.25 and 0.5 mM target metal ion concentrations and a ﬁnal BSA concentration of 0.25 mM. The mixtures were incubated at room temperature for 2 h to allow complex formation between the metal ions and test protein. IR spectra of the pure protein solution and every solution of the metal ion–protein mixture were collected using hydrated ﬁlms over a transmittance range of 4000–400 cm−1 at a resolution of 4 cm−1 and 100 scans. The difference spectrum was produced by subtracting the spectra of the BSA solution from that of the metal ion–BSA complex by following the method described by Dousseau et al., 1989 [20]. Sci. Pharm. 2019, 87, 5 5 of 13

2.4. Analysis of Protein Conformation Secondary structures of the test protein, before and after interaction with each selected metal ion, were evaluated over a transmittance range of 1700–1600 cm−1 by following the procedure of Byler and Susi, (1986) [21]. Intensity variations and the spectral shifting of the BSA amide A band (NH stretching) at 3500 cm−1, amide I band (C=O stretch.) at 1700–1600 cm−1 and the amide II band (C–N stretch. coupled to NH bending) at 1550 cm−1 were monitored after interactions with selected metal ions. For analysis of the secondary structure of the protein, four major peaks were resolved and characterized. The above-mentioned spectral regions were deconvolved by a curve ﬁtting method using Origin 2018 Graphing and Analysis software. These peaks, characteristic to an α-helix (1660–1650 cm−1), β-sheet (1637–1614 cm−1), β-turn (1678–1670 cm−1), and a β-antiparallel (1691–1680 cm−1), were corrected and the corresponding areas were calculated with the help of Gaussian functions. The area of the respective bands, characteristic to amide I components, was summed-up and divided by the total area to obtain the percentage area of the amide I components [15,22,23].

3. Results and Discussion The interaction of bovine serum albumin, a major plasma protein, with a variety of metal ions including group A metal ions (Ca2+ and Ba2+), noble metal ions (Ag+ and Ru3+) and heavy metal ions (Cu2+ and Co2+) was estimated by using FT-IR spectroscopy and its derivative methods. The shifting of the spectral signals and the variation in the dimension of the amide I band of the protein at 1700–1600 cm−1 (C=O stretching), amide II band near 1550 cm−1 (C–N stretch. coupled with N–H bending), and amide A band near 3500 cm−1 (NH stretch.) were monitored upon metal ion interaction [21,23]. The backbone conformation of the protein is directly related to the amide I band, while the shape of the amide I band is characteristic to the secondary structures as an α-helix (1660–1650 cm−1), β-sheet (1637–1614 cm−1), β-turn (1678–1670 cm−1) and β-antiparallel (1691–1680 cm−1). The difference spectra were produced by subtracting the spectra of the free protein from that of the BSA–metal ion complexes. These difference spectra were used for monitoring the intensity variations upon complex formation. In order to determine the secondary structures of the protein, self-deconvolution and curve ﬁtting methods were applied [21]. The secondary structures of the free protein and the protein–metal ion complexes were analyzed and the percentages of α-helix, β-sheet, β-turn and β-antiparallel were calculated to measure the changes on complex formation. The second derivative resolution enhancement curves were obtained for free BSA and its complexes in the range of 1700–1600 cm−1. The intensity and total area of the peaks were calculated in the corresponding range of the different structural components of the protein.

3.1. BSA and Group A Metal Ion (Ca2+ and Ba2+) Interactions Ca2+ and Ba2+ were allowed to interact with BSA at two different concentrations, whereas the protein concentration was kept constant in the sample solutions. At low metal ion concentrations (0.125 mM), the intensity of the BSA amide I peak (1651 cm−1, free BSA) and amide II peak (1545 cm−1, free BSA) was slightly reduced in the difference spectra of BSA–Ca2+ and BSA–Ba2+ complexes, while at a higher metal ion concentration (0.5 mM), the spectral intensities of the above bands were found to be signiﬁcantly decreased. In the difference spectrum, strong negative features for the amide I and amide II bands were seen. These bands were at 1653 cm−1 (negative band) and 1538 cm−1 (positive band), respectively, for the Ca2+–BSA complex, whereas, in the case of the Ba2+–BSA complex, the bands were observed at 1654 cm−1 (negative band) and 1536 cm−1 (positive band), respectively (Figure2A,B). These reductions in the intensities of bands have indicated the interactions of Ca2+ and Ba2+ with the C=O, COO− and NH groups of BSA as per the HSAB Lewis theory of binding, as shown in Table1. Sci. Pharm. 2019, 87, 5 6 of 13 Sci. Pharm. 2019, 87, x FOR PEER REVIEW 6 of 13

Figure 2. Representative FT-IR spectra; (A): free BSA, the BSA–Ca2+ complex and its difference spectra; 2+ Figure(B): free 2. BSA, Representative the BSA–Ba FT2+-IRcomplex spectra and; (A) its: free difference BSA, the spectra. BSA–Ca complex and its difference spectra; (B): free BSA, the BSA–Ba2+ complex and its difference spectra. The binding interactions of Ca2+ and Ba2+ with the BSA protein were further revealed by the bandThe shifting binding of theinteractions amide A of band Ca2+ and from Ba 34392+ with cm the−1 (N–HBSA protein stretch.) were for further free BSA revealed to 3445 by andthe band 3450 shiftingcm−1 in of the the BSA–Ca amide2+ Aand band BSA–Ba from 34392+ complexes, cm−1 (N–H respectively, stretch.) for as free evident BSA to from 3445 Figure and 23450A,B. cm The−1 in band the BSAshifting–Ca2 towards+ and BSA a higher–Ba2+ complexes, frequency wasrespectively, probably as due evident to the from interaction Figure of 2A the,B.Ca The2+ and band Ba shifting2+ ions withtowards the N–Ha higher and frequency C–N groups was of probably bovine serum due to albumin. the interaction Furthermore, of the Ca spectral2+ and shiftingBa2+ ions was with also the seen N– Hfor and the C amide–N groups I band of from bovine 1651 serum cm−1 albumin.(free BSA) Furthermore, to 1653 cm− spectral1 (Ca2+–BSA shifting complex) was also and seen 1654 for cm the−1 amide(Ba2+–BSA I band complex). from 1651 This cm shifting−1 (free ofBSA) the to amide 1653 Icm band−1 (Ca was2+–BSA due tocomplex) the interactions and 1654 of cm Ca−12+ (Baand2+–BSA Ba2+ complex).ions with theThis C–N shifting and C–Oof the groups amide of I band the protein, was due whereas to the interactions the reduction of in Ca the2+ and intensity Ba2+ ions of the with amide the CI band–N and in C the–O spectragroups ofof BSA–Cathe protein,2+ and whereas BSA–Ba the2+ reductionat 1651cm in −the1 suggested intensity of a the signiﬁcant amide I decreaseband in the of spectraα–helix ofin theBSA secondary–Ca2+ and protein BSA–Ba structure2+ at 1651 at relativelycm−1 suggested higher a metal significant concentrations decrease [ 24of]. α The–helix bidentate in the secondarycoordination protein of the structure COO- group at relatively of BSA was higher also metal evident concentrations upon the interaction [24]. The of b theidentate Ca2+ andcoordination Ba2+ ions - – −1 2+ −12+ 2+ ofas athe downshift COO group position of BSA of the wasνas also(COO evident) band upon from the 1367 interaction cm (free of BSA) the toCa 1364 and cm Ba (Ca ions–BSA as a downshiftcomplex) and position 1363 cmof the−1 (Baνas (COO2+–BSA--) ba complex)nd from was 1367 observed. cm−1 (free BSA) to 1364 cm−1 (Ca2+–BSA complex) and 1363The cm quantitative−1 (Ba2+–BSA estimation complex) ofwas the observed. secondary structure of free BSA and the Ca2+–BSA and Ba2+–BSA complexes was carried out in the spectral range of 1700–1600 cm−1 and the conformational 2+ 2+ changesThe afterquantitative protein–metal estimation ions interactionsof the secondary were structure evaluated. of In free agreement BSA and with the Ca the reported–BSA and values, Ba – −1 BSAthe secondary complexes structure was carried of free out BSA in the was spectral found torange consist of 1700 of an–1600α-helix cm (1651 and cm the− 1 conformational; 62%), β-turn changes(1673 cm after−1; 14%), proteinβ-sheet–metal (1614, ions interactions 1628 cm−1; 22%)were andevaluated.β-antiparallel In agreement (1681 cmwith−1 ;the 2%) reported [15]. However, values, −1 the secondary binding interaction structure ofof BSAfree BSA with was the Cafound2+ and to consist Ba2+ ions of an resulted α-helix in (1651 a signiﬁcant cm ; 62%), reduction β-turn in(1673 the −1 −1 −1 cmα-helix; 14%), from β 62%-sheet for (1614, free BSA 1628 to 50% cm ; for 22%) the Ca and2+ –BSA β-antiparallel complex (1681(1645, cm1650,; and 2%) 1660[15]. cm How−1)ever, and 46%the 2+ 2+ bindingfor the Ba interaction2+–BSA complex of BSA (1656with cmthe− Ca1), with and a Ba considerable ions resulted increase in a insignificant the β-sheet reduction from 22% in inthe free α- 2+ −1 helixBSA tofrom 29% 62% in thefor free BSA–Ca BSA2+ tocomplex 50% for the (1624 Ca cm–BSA−1) andcomplex 39% (1645, in the 1650, BSA–Ba and2+ 1660complex cm ) (1633and 46% cm −for1). 2+ −1 theThe Ba results–BSA of complex the protein’s (1656 secondary cm ), with structure a considerable analysis increase are depicted in the in β Table-sheet2 fromand Figure 22% in3A,E,G. free BSA to 29% in the BSA–Ca2+ complex (1624 cm−1) and 39% in the BSA–Ba2+ complex (1633 cm−1). The results of the protein’s secondary structure analysis are depicted in Table 2 and Figure 3A,E,G.

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Table 2. Secondary structure analysis for free BSA and the Ca2+, Ba2+, Ag+, Ru3+, Cu2+ and Co2+ complexes at 0.5 mM metal ion concentration.

Free BSA BSA–Cu2+ BSA–Ca2+ BSA–Ag+ BSA–Ba2+ BSA–Co2+ BSA–Ru3+ Amide I Components (%) Complex (%) Complex (%) Complex (%) Complex (%) Complex (%) Complex (%) β-sheet (± 2) 1614–1637 cm−1 22 34 29 31 39 29 31 α-helix (± 4) 1650–1660 cm−1 62 48 50 52 46 37 54 β-turn (± 2) 1670–1678 cm−1 14 18 21 17 15 30 15 β-antiparallel (± 1) 1680–1691 cm−1 2 0 0 0 0 4 0 Sci. Pharm. 2019, 87, 5 8 of 13 Sci. Pharm. 2019, 87, x FOR PEER REVIEW 8 of 13

Figure 3. 3. SecondSecond-derivative-derivative resolution resolution enhancement enhancement and and curve curve-ﬁtted-fitted amide amide I band I bandbetween between the 1700 the– −1 1700–16001600 cm−1 cmregionregion for free for BSA free BSA(0.25 (0.25 mM) mM) and and its itscomplexes complexes with with selected selected metal metal ions ions (metal (metal ions 2+ + 2+ concentration == 0.5 mM).mM). ((AA):): FreeFree BSA;BSA; ((BB):): BSA-CoBSA-Co2+ complex; ((C):): BSA-AgBSA-Ag+ complex; (D): BSA-CuBSA-Cu2+ 2+ 3+ 2+ complex; (E): BSA-CaBSA-Ca2+ complex;complex; ( (FF):): BSA BSA-Ru-Ru3+ complex;complex; (G (G):): BSA BSA-Ba-Ba2+ complexcomplex.. 3.2. BSA and Noble Metal Ion (Ag+ and Ru3+) Interactions 3.2. BSA and Noble Metal Ion (Ag+ and Ru3+) Interactions Similar to the BSA–Ca2+ and BSA–Ba2+ interactions, the intensities of the amide I and amide II bandsSimilar (1651 andto the 1545 BSA cm–Ca−12respectively;+ and BSA–Ba free2+ interactions, BSA) were signiﬁcantlythe intensities reduced of the amide due to I the and interaction amide II bands (1651 and 1545 cm−1 respectively; free BSA) were significantly reduced due to the interaction of the protein with the Ag+ and Ru3+ ions. Significant reduction in the band intensities was found at

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higher metal ion concentrations (0.5 mM), with strong negative features in the difference spectrum. The amide I and amide II bands of the BSA–Ag+ complex were observed at 1649 cm−1 (negative band) and 1544 cm−1 (negative band), respectively, whereas, for the BSA–Ru3+ complex, they were found to be at 1656 cm−1 (negative band) and 1544 cm−1 (negative band), respectively (Figure 4A,B). The variation in the band intensities was due the complexation of the Ag+ and Ru3+ ions at the C=O, COO− and NH groups of the protein. It was noticed that the C=O stretching vibration of free BSA was influenced by the interaction of the protein with the Ag+ and Ru3+ ions, whereas the C–N stretching and bending vibrations were less affected by these metal ions, indicating an insignificant effect on the amide II bands. In addition to band intensity variations, spectral shifting was also detected, as the Sci. Pharm. 2019, 87, 5 9 of 13 amide I band of free BSA at 1651 cm−1 was shifted to 1649 and 1655 cm−1 for the sample solutions containing BSA–Ag+ and BSA–Ru3+ mixtures, respectively. This shifting of the amide I band was due ofto thethe proteininteractions with of the the Ag Ag+ and+ and Ru Ru3+3+ions. ions Significantwith the C=O reduction groups inof thethe bandprotein. intensities was found at higher metal ion concentrations (0.5 mM), with strong negative features in the difference spectrum. The amideThe results I and amide of the IIquantitative bands of the determination BSA–Ag+ complex of the were secondary observed protein at 1649 structure cm−1 (negative and its band)complexes and 1544 with cm Ag−+1 (negativeand Ru3+ band),ions, over respectively, the spectral whereas, region for of the 1700 BSA–Ru–16003+ cmcomplex,−1, have theyindicated were foundsignificant to be interactions at 1656 cm− 1 between(negative the band) protein and 1544 and cmmetal−1 (negative ions. The band), α-helix respectively in free BSA (Figure (62%)4A,B). was Theremarkably variation reduced in the band to 52% intensities and 54% was in the due BSA the– complexationAg+ (1650 and of 1660 the cm Ag−+1)and and RuBSA3+ –ionsRu3+ at complexes the C=O, COO(1651− andand 1660 NH groupscm−1), respectively. of the protein. By It contrast was noticed, a significant that the C=O increase stretching in the vibrationβ-sheet from of free 22% BSA in wasfree influencedBSA to 31% by in the the interaction BSA–Ag+ of(1616 the and protein 1631 with cm− the1) complex Ag+ and as Ru well3+ ions, as the whereas BSA–Ru the3+ C–N(1616 stretching and 1631 andcm−1 bending) complex vibrations, with a relatively were less lesser affected change by these in the metal β-turn ions, from indicating 14% in anfree insignificant BSA (1673 cm effect−1) to on 17% the amidein the BSA II bands.–Ag+ Incomplex addition (1678 to bandcm−1) intensityand 15% variations,in the BSA spectral–Ru3+ complex shifting (1678 was cm also−1) detected,, was observed. as the amideInterestingly, I band the of free β-antiparallel BSA at 1651 (1681 cm− cm1 was−1; 2% shifted in free to BSA) 1649 was and absent 1655 cmin both−1 for the the BSA sample–Ag+ and solutions BSA– containingRu3+ complexes BSA–Ag (1691+ and–1680 BSA–Ru cm−1, 3+0%)mixtures, (Table 2; respectively. Figure 3A,C, ThisF). shiftingThe decrease of the amidein the Iintensity band was of duethe toamide the interactions I band was ofdue the to Ag the+ reductionand Ru3+ ionsof the with α–helix the C=O in the groups secondary of the structure protein. of the protein [24].

Figure 4. Representative FT-IR spectra; (A): free BSA, the BSA–Ag+ complex and its difference spectra; Figure 4. Representative FT-IR spectra; (A): free BSA, the BSA–Ag+ complex and its difference spectra; (B): free BSA, the BSA–Ru3+ complex and its difference spectra. (B): free BSA, the BSA–Ru3+ complex and its difference spectra. The results of the quantitative determination of the secondary protein structure and its complexes 3.3. BSA and Heavy Metal Ion (Cu2+ and Co2+) Interactions with Ag+ and Ru3+ ions, over the spectral region of 1700–1600 cm−1, have indicated signiﬁcant interactions between the protein and metal ions. The α-helix in free BSA (62%) was remarkably An insignificant reduction in the peak intensities was found for the amide I band (1651 cm−1, free reduced to 52% and 54% in the BSA–Ag+ (1650 and 1660 cm−1) and BSA–Ru3+ complexes (1651 and BSA) and amide II band (1545 cm−1, free BSA) in the difference spectra of the BSA–Cu2+ and BSA–Co2+ 1660 cm−1), respectively. By contrast, a signiﬁcant increase in the β-sheet from 22% in free BSA to complexes at lower metal ion concentrations (0.125 mM). By contrast, at 0.5 mM metal ion 31% in the BSA–Ag+ (1616 and 1631 cm−1) complex as well as the BSA–Ru3+ (1616 and 1631 cm−1) concentration, the intensities of the amide I and amide II bands were significantly decreased with complex, with a relatively lesser change in the β-turn from 14% in free BSA (1673 cm−1) to 17% in remarkable negative characteristics in the difference spectra. The amide I and amide II bands of the the BSA–Ag+ complex (1678 cm−1) and 15% in the BSA–Ru3+ complex (1678 cm−1), was observed. BSA–Cu2+ complex were at 1650 cm−1 (negative band) and 1544 cm−1 (positive band), respectively, Interestingly, the β-antiparallel (1681 cm−1; 2% in free BSA) was absent in both the BSA–Ag+ and

BSA–Ru3+ complexes (1691–1680 cm−1, 0%) (Table2; Figure3A,C,F). The decrease in the intensity of the amide I band was due to the reduction of the α–helix in the secondary structure of the protein [24].

3.3. BSA and Heavy Metal Ion (Cu2+ and Co2+) Interactions An insigniﬁcant reduction in the peak intensities was found for the amide I band (1651 cm−1, free BSA) and amide II band (1545 cm−1, free BSA) in the difference spectra of the BSA–Cu2+ and BSA–Co2+ complexes at lower metal ion concentrations (0.125 mM). By contrast, at 0.5 mM metal ion concentration, the intensities of the amide I and amide II bands were signiﬁcantly decreased with Sci. Pharm. 2019, 87, 5 10 of 13 remarkable negative characteristics in the difference spectra. The amide I and amide II bands of the 2+ −1 −1 Sci.BSA–Cu Pharm. 2019complex, 87, x FOR were PEER atREVIEW 1650 cm (negative band) and 1544 cm (positive band), respectively,10 of 13 while these bands were at 1654 cm−1 (negative band) and 1547 cm−1 (negative band) for the BSA–Co2+ whilecomplex these (Figure bands5 A,B).were at The 1654 reduction cm−1 (negative of the intensitiesband) and was1547 duecm−1 to(negative the interaction band) for of the heavy BSA metal–Co2+ complexions with (Figure the C=O, 5A,B C–N). The and reduction NH groups of the of intensities BSA as per was the due HSAB to the Lewis interaction theory of heavy binding metal (Table ions1). withThe bindingthe C=O, interactions C–N and NH between groups the of BSA test proteinas per the and HSAB the metal Lewis ions theory (Cu 2+of andbinding Co2+ ()Table were 1). further The bindingevidenced interactions by the spectral between shifting the oftest the protein amide and I band the from metal 1651 ions cm (Cu−1 in2+ freeand BSA Co2+ to) were 1655 cm further−1 in evidencedthe BSA–Cu by2+ theand spectral 1654 cm shifting−1 in the of BSA–Cothe amide2+ Icomplexes. band from This1651 spectral cm−1 in shiftingfree BSA was to 1655 reported cm−1 in due the to BSAthe interactions–Cu2+ and 1654 of the cm Cu−1 in2+ theand BSA Co2+–Coions2+ complexe with thes. C=O This and spectral C–N groupsshifting of was the reported protein. Indue addition, to the interactionsthe shifting of thethe proteinCu2+ and amide Co2+ ions A band with from the 3439C=O cmand−1 Cto–N 3445 groups and 3443of the cm protein.−1 in the In BSA–Cu addition,2+ andthe shiftingBSA–Co of2+ thecomplexes, protein amide respectively, A band has from supported 3439 cm the−1 to evidence 3445 and of 3443 metal cm ion−1 in interactions the BSA–Cu with2+ and the BSA C–N– Coand2+ complexes N–H groups, respectively of bovine serum, has supported albumin (Figure the evidence5A,B). of metal ion interactions with the C–N and N–H groups of bovine serum albumin (Figure 5A,B).

FigureFigure 5. 5. RepresentativeRepresentative FT FT-IR-IR spectra spectra;; ( (AA)):: free free B BSA,SA, BSA BSA–Cu–Cu22++ complexcomplex and and its its difference difference spectra spectra;; ((BB)):: free free B BSA,SA, BSA BSA–Co–Co22++ complexcomplex and and its its difference difference spectra spectra..

TheThe q quantitativeuantitative analysis analysis of of the the secondary secondary protein protein structures structures revealed revealed that that the the interaction interaction of of the the 2+ 2+ CuCu2+ andand Co Co2+ ionsions with with the the test test protein protein has has resulted resulted in a in major a major decrease decrease in the in theproportion proportion of the of theα- α 2+ −1 helix-helix structure structure of offree free BSA BSA (62%) (62%) to to 48% 48% in in the the BSA BSA–Cu–Cu2+ complexcomplex (1647, (1647, 1660 1660 cm cm−1) )and and 37% 37% in in the the 2+ −1 β BSABSA–Co–Co2+ complexcomplex (1650 (1650 cm cm−1). On). Onthe other the other hand, hand, there there was a was significant a significant increase increase in the in β- thesheet-sheet from 2+ −1 2+ 22%from for 22% free for BSA free BSA to 34% to 34% for forthe theBSA BSA–Cu–Cu2+ complexcomplex (1616, (1616, 1632 1632 cm cm−1) and) and 29% 29% for for the the BSA BSA–Co–Co2+ −1 β complexcomplex (1614, (1614, 1627 1627 cm cm−1).). The The proportion proportion of of the the β-turn-turn was was also also considerably considerably increased increased due due to to metal metal 2+ −1 ionion–protein–protein interactions interactions and and it itwas was found found to tobe be18% 18% in the in theBSA BSA–Cu–Cu2+ (1678(1678 cm−1) cm and 30%) and in 30% the BSA in the– 2+ −1 CoBSA–Co2+ (1670 cm(1670−1) complexes cm ) complexes in comparison in comparison to 14% topresent 14% present in free inprotein. free protein. The reduction The reduction in the inband the intensitiesband intensities was probably was probably due to due the toquantitative the quantitative variation variation in the inprotein’s the protein’s secondary secondary structure structure.. The 2+ 2+ rTheesults results of the of protein’s the protein’s secondary secondary structure structure estimation estimation after after interaction interaction with with Cu2+ Cu and andCo2+ Co ions ionsare presentedare presented in Table in Table 2 and2 and Figure Figure 3A,3B,A,B,D.D. 3.4. Comparison of the Interaction Results with Our Previous Study Using Affinity Capillary Electrophoresis 3.4.(ACE) Comparison of the Interaction Results with Our Previous Study Using Affinity Capillary Electrophoresis (ACE) The metal ion–protein interaction results obtained in this study were compared with our previous report,The where metal we ion had–protein investigated interaction the bindingresults obtained interactions in this between study a were number compared of metal with ions ourand previousbiologically report significant, where we proteins had investigated including the BSA binding using affinityinteraction capillarys between electrophoresis a number of [metal10]. In ions the andprevious biologically study, significant the intensity proteins of the including interaction BSA was using represented affinity bycapillary the extent electrophoresis of the mobility [10]. shift In the of previous study, the intensity of the interaction was represented by the extent of the mobility shift of analyte proteins in the capillary after interaction with the metal ions. The variation in the mobility shift in ACE was compared with the percent change in the α-helical structure of BSA in this study after interaction with the metal ions. This percent change in the α-helical structure in the secondary structure of the protein was calculated by Equation (1):

Sci. Pharm. 2019, 87, 5 11 of 13 analyte proteins in the capillary after interaction with the metal ions. The variation in the mobility shift in ACE was compared with the percent change in the α-helical structure of BSA in this study after interaction with the metal ions. This percent change in the α-helical structure in the secondary structure of the protein was calculated by Equation (1):

% change in α-helix = (% α-helix in metal ion-BSA complex- % α-helix in free BSA)/ (1) (% α-helix in free BSA) × 100

The comparison of the interaction results obtained from FT-IR and ACE analysis is summarized in Table3. It is evident from the table that the mobility shift (given as ∆R/Rf ± cnf ) obtained in the ACE analysis due to the metal ion–BSA interactions was comparable to the percent change in the α-helical structure of the protein as determined by FT-IR analysis. In both the techniques, similar interaction intensities were obtained, with Co2+ ions exhibiting the highest binding afﬁnity towards BSA, followed by Ba2+ ions. However, small differences in the results of the binding interactions of Ag+ and Ru3+ to BSA were observed between the two techniques. Ag+ exhibited the least afﬁnity towards BSA in the ACE analysis, while Ru3+ showed minimum interaction with the protein when investigated with the FT-IR technique. The similar interaction results proved that both the techniques can be utilized to study the metal ion–protein interactions with accurate binding results. However, the FT-IR technique is supposed to be advantageous due to its simple analytical procedure, rapid analysis and cost effectiveness.

Table 3. Comparison of the BSA–metal ion interaction results obtained in this study with those reported in our previous investigation using Afﬁnity Capillary Electrophoresis.

% Variation in α-Helix after ± 1 Metal Ions ∆R/Rf cnf Values in ACE [10] Complexation (This Study) Co2+ −0.0482 ± 0.0089 −40.3 Ba2+ −0.0434 ± 0.0025 −25.8 Cu2+ −0.0420 ± 0.0038 −22.58 Ca2+ −0.0402 ± 0.0115 −19.35 Ru3+ −0.0142 ± 0.0053 −12.9 Ag+ 0.0053 ± 0.0056 −16.12 1∆R: difference between the mobility ratios of BSA with and without metal ions in the capillary. Rf: the mobility ratio of BSA without metal ion interaction. Cnf : conﬁdence interval calculated for six replicate runs.

4. Conclusions In the event of increased drug resistance to traditional medicines, much research is being carried out into developing new agents to overcome these problems. Recently, metal-based complexes have emerged as promising agents with reported antiviral, antimicrobial and anticancer potential. In order to design metallodrugs, a crucial step is to analyze the ability of biologically active metal ions to bind with metalloproteins such as albumin, as it plays important roles in the transportation and distribution of these metal ions. The current research was aimed at delivering an approach to study the interactions of Ca2+, Ba2+, Ag+, Ru3+, Cu2+ and Co2+ ions with the BSA protein at physiological pH using the FT-IR spectroscopic technique. The binding interactions of the selected metal ions were demonstrated by significant variations in the intensities of the amide I band of the BSA after metal complexation. The binding interaction was further revealed by the spectral shifting of the amide I, amide II and amide A bands. The intensity variations and spectral shifting were due to the interaction of the metal ions with the O and N atoms of the polypeptide chain. The binding affinities of the selected metal ions were found to be in the following order: Co2+ > Ba2+ > Cu2+ > Ca2+ > Ag+ > Ru3+. The interactions of the metal ions with the protein have resulted in the alteration of the protein’s secondary structure, which was characterized by a remarkable reduction in the α-helical structure of BSA. The reduction in the α-helix proportion was due to its conversion to a β-sheet and β-turn, Sci. Pharm. 2019, 87, 5 12 of 13 resulting in a partial destabilization of the protein. All the prominent interactions were effectively supported by the HSAB Lewis theory of metal ions and ligand binding. The present findings set a benchmark for further investigations on protein–metal ion interactions using FT-IR and are useful in clinical and pharmaceutical research.

Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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