BIBECHANA Vol. 21, No. 3, December 2024, 311-320 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher: Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University) Biratnagar Experimental FTIR characterization of kidney stones, DFT analysis of CaC2O4 and its interactions with lysozyme Arjun Acharya1, Madan Khanal1, Rajesh Maharjan1, Kalpana Gyawali1, Kamal Khanal1, Mohan Bahadur Kshetri1, Bhoj Raj Luitel2, Rameshwar Adhikari3,4, Deependra Das Mulmi5, Tika Ram Lamichhane1,∗, Hari Prasad Lamichhane1, 1Central Department of Physics, Tribhuvan University, Kathmandu 44600, Nepal 2Department of Urology and Kidney Transplant Surgery, Tribhuvan University , Teaching Hospital, Institute of Medicine, Maharajgung, Kathmandu 44600,Nepal 3Central Department of Chemistry, Tribhuvan University, Kathmandu 44600, Nepal 4Research Center for Applied Science and Technology (RECAST), Tribhuvan University, Kathmandu 44600, Nepal 5Nanomaterials Research Laboratory, Nepal Academy of Science and Technology, Lalitpur 44700, Nepal ∗Corresponding author: Email: tika.lamichhane@cdp.tu.edu.np Abstract Kidney stone is an alarming global disease due to its rising incidence and prevalence. FTIR spectroscopic analysis reveals that calcium oxalate is one of the most frequent chemical constituents in kidney stones. DFT calculations indicate that the calcium oxalate can interact through charge transfer process in biological activities. Among various proteins, lysozyme is one of the promoter proteins in nephrolithiasis of calcium oxalate type kidney stone. The location, conformation and interactions of calcium oxalate with the active residues of lysozyme contribute the binding energy of -4.18 kcal/mol. The characterization of kidney stones, DFT calculations of calcium oxalate, and binding interactions of calcium oxalate-lysozyme complex contribute to the understanding of nephrolithiasis. Keywords Kidney stone; Calcium oxalate; Lysozyme; Fourier transform infrared spectroscopy; Density func- tional theory; Molecular docking; Nephrolithiasis. Article information Manuscript received: August 14, 2024; Revised: September 25, 2024; Accepted: September 26, 2024 DOI https://doi.org/10.3126/bibechana.v21i3.68781 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 311 http://nepjol.info/index.php/BIBECHANA tika.lamichhane@cdp.tu.edu.np https://doi.org/10.3126/bibechana.v21i3.68781 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 312 1 Introduction Kidney stone is one of the oldest diseases, charac- terized by the formation of solid masses within the urinary system. These deposits are strongly linked to chronic kidney disease and can lead to kidney failure [1, 2]. Despite increased awareness and im- proved treatment procedures, the prevalence of kid- ney stone disease is increasing globally, presenting a significant health threat [3]. The prevalence ranges from 7% to 13% in North America, 5% to 9% in Eu- rope, and 1% to 5% in Asian countries [4]. In Asia, the western, southern, and southwestern regions ex- hibit particularly high rates, with prevalence rang- ing from 5% to 19.1%. In contrast, East Asian and Northern Asian regions show a lower prevalence, ranging from 1% to 8% [5]. The study in Kathmandu University Teaching Hospital about the distribution of urinary calculi based on age, gender and types shows 54.20% cases are of 21-40 years. Among the total kid- ney stone patients, male population (57.95%) are more prone than the female population (42.05%). Again, 74.16% of the total patients are suffered from calcium oxalate and calcium phosphate type urinary calculi. Non-vegetarian groups (88.70%) are found more suffered from this disease than veg- etarian groups (11.30%). Further, this study also suggested spectroscopic analysis of urinary calculi [6]. Computed tomography (CT) urographic study in Lumbini Medical College, Palpa, Nepal shows 76.7% of urinary obstruction are due to urinary calculi. Among the patients, 60.7% are male and 39.3% are female, and 46.7% of patients lie within 21-40 years [7]. In another research of urinary cal- culi in Kathmandu Pathology Laboratory using bio- chemical techniques shows calcium oxalate as a ma- jor constituents with male to female patients ratio 1: 1.2 [8]. In a study of urinary calculi patients in Shree Birendra Hospital, Kathmandu, Nepal, it is found that 69.2 % of the affected population are male and 30.8% are female. The majority of patients, 51%, fall within the age range of 31-45 years. Additionally, calcium oxalate is found in 64.25% of the cases. Among the ethnic groups, maximum number (30.75%) of the patients are from the Janajati community, while minimum (11.25%) are from the Madhesi population. Geographically, 85.7% of the cases are reported in the Hilly region, with 14.3% originating from the Terai/Madhesh re- gion [9]. Kidney stones are generally developed from cal- cium oxalate, calcium phosphate, magnesium am- monium phosphate, uric acid, and cystine. Among all, calcium oxalate (CaC2O4) is the most fre- quently observed in the kidney stones [10]. Fourier transform infrared (FTIR) spectroscopic technique is a powerful analytical tool for the prediction of chemicals in the samples through the measurement of wide range of spectra. It has high sensitivity, quantitative accuracy, and superior signal-to-noise ratio [11] and is widely used in the characterization of different types of kidney stones [12, 13]. Den- sity functional theory (DFT) analysis can be em- ployed to study the dipole moment, frontier molec- ular orbitals, global reactive descriptors, molecular electrostatic potential (MEP), and Mulliken atomic charges. These quantum mechanical properties pro- vide insights about the potential interactions be- tween chemical compound and protein, as explained in our previous work [14,15]. Different proteins contribute as promoter in the biomineralization. In kinetic studies of calcium ox- alate type crystal growth in the presence of pro- teins lactoferrin and lysozyme revealed that both the proteins promote crystal growth, with lysozyme exhibiting a more effective role in accelerating the growth process [16]. In our previous work, lacto- ferrin protein has exhibited the binding efficacy of -3.86 kcal/mol [14] and this research focuses on studying the different interactions by active amino acids of lysozyme protein and their contribution for binding efficacy with calcium oxalate at the atomic level. Molecular docking analysis of protein and lig- and can predict the different conformation of lig- and within the selected binding pocket of target protein. It also predicts the binding affinities using different algorithms and scoring function [17]. The molecular docking of ligand with matrix proteins of kidney stones is found to be significant for studying the calcium oxalate types nephrolithiasis [18]. Such studies at the atomic level will be helpful for under- standing the interactions between calcium oxalate and lysozyme. Current research aims to characterize the kid- ney stones. Further, this research will study the quantum mechanical properties of calcium oxalate using DFT in water solvent, along with the inter- actions within binding domain of promoter protein, lysozyme. This study will contribute to the un- derstanding of globally challenged and unclear phe- nomena of nephrolithiasis. 2 Methodology 2.1 Characterization of samples Kidney stones were collected from the institute of medicine, Maharajgung following the receipt of ap- proval from the Institutional Review Committee of the Institute of Medicine, Tribhuvan University, Nepal. Informed consents were taken from all re- spondents for the analysis of samples. The col- lected stones were carefully cleaned with distilled water and air-dried at room temperature, then Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 313 crushed using mortar and pestle. Finally, samples were characterized under FTIR spectroscopy using the SHIMADZU IRAffinity-1S spectrophotometer within the range of 400-4000 cm−1. 2.2 Quantum mechanical calculations Three dimensional structure of calcium oxalate (ID: 33005) was obtained from PubChem, an open chemistry database at the National Institutes of Health, USA [19], and optimized using the DFT at B3LYP/6-311++G(d,p) level of calculation in the water solvent using the integral equation formula- tion of the polarizable continuum model (IEFPCM) in Gaussian 16W software [20]. Using the optimized structure, dipole moment, Mulliken charges, molec- ular electrostatic potential (MEP), highest occu- pied molecular orbital (HOMO) and lowest unoc- cupied molecular orbital (LUMO), and density of states (DOS) were calculated. The energies of HOMO and LUMO orbitals, ion- ization potential and electron affinity of chemical compound were calculated using Koopmans’ theo- rem as [21]: Ionization potential, I = −EHOMO (1) Electron affinity, A = −ELUMO (2) The global reactive descriptors (chemical hard- ness, chemical softness, electronic chemical poten- tial, and global electrophilicity index) were calcu- lated using ionization potential and electron affinity as follows [21–23]: Chemical hardness: η = I −A 2 (3) Chemical softness: S = 1 η (4) Electronic chemical potential: µ = −I +A 2 (5) Global electrophilicity index: ω = µ2 2η (6) Additionally, ultraviolet-visible (UV-Vis) spec- tra were generated using the time-dependent (TD)- DFT method at the same level of calculation in water solvent. The compound was visualized and evaluated using GaussView 6 [24] and GaussSum 3.0 [25] software programs. 2.3 Molecular docking The structure of human lysozyme (7xf6.pdb) was downloaded from the data center for the global pro- tein data bank, RCSB PDB [26]. The secondary structures were analyzed by Ramachandran plot us- ing Discovery Studio Visualizer v21.1.0.20298 [27]. The surface area, volume, and active residues in the binding region were predicted using the com- puted atlas of surface topography of the universe of proteins (CASTp) open-access online server [28]. The PDB files of protein and ligand were converted into partial charge and atom type (PDBQT) format after removal of water molecules, addition of Koll- mann’s charges, and integration of polar hydrogen atoms using the AutoDockTools [29–31]. Molecu- lar docking using AutoDock4 [29] was carried out within the grid points (40× 40 × 40) of grid point spacing 0.375 Å along the x, y, and z axes, re- spectively. All the molecular docking procedures were conducted as described in our previous studies [14, 32]. Finally, the best conformation of protein- ligand complex was generated using the graphical user interface of AutoDockTools. The visualization of protein-ligand complexes were carried out using PyMOL 2.5.2 [33] and LigPlot+ v.2.2 [34]. 3 Results and discussion 3.1 Experimental FTIR characterization of kidney stones FTIR spectroscopic analysis was carried out for forty-four kidney stones. Comparing the generated spectra with literature [12,13], the samples contains whewellite, struvite, and uric acid types chemicals in pure and mixed form (Figure 1). The spectra of majority of the samples show intense peaks at 1608-1613 cm−1 and 1313-1315 cm−1 with less in- tense peak at 1380-1388 cm−1 (Figure 2) and these peaks almost match with the calcium oxalate mono- hydrate (whewellite) type kidney stones [12]. In our previous work, FTIR spectra of calcium oxalate generated in different solvent using DFT method at the B3LYP/6-311++G(d,p) level of calculation, the spectra in water solvent was closely matched with the experimental FTIR spectra of whewellite [14]. Figure 1: Pie chart showing the distribution of chemical compositions in the samples analyzed by Fourier transform infrared spectroscopic technique. Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 314 Figure 2: Experimental Fourier transform infrared spectra of the thirty-one whewellite type kidney stone samples. 3.2 Quantum mechanical calculation of calcium oxalate 3.2.1 Geometry optimization The optimized structure of calcium oxalate is shown in Figure 3. In the present study, calculated dipole moment in water solvent is found 24.28 Debye, whereas in our previous work in gaseous phase, it was 14.88 Debye [14]. The dipole-dipole inter- actions between ligand and protein can affect the binding energy of the protein-ligand complex [35]. Thus, calcium oxalate can also interact through dipole-dipole interactions with the nephrolithiatic proteins. Figure 3: Optimized structures of CaC2O4 by the DFT method at the B3LYP/6-311++G(d,p) level of calculation in water solvent. 3.2.2 Frontier molecular orbitals In the electronic absorption spectra of calcium ox- alate in water solvent, the wavelengths of 278 nm, 265 nm, and 231 nm are responsible for the elec- tronic transitions (Table 1). The maximum oscil- latory strength is observed for wavelength 231 nm which is due to major contribution from the HOMO to LUMO+1 transition of electrons (Figure 4a and Table 1). The electronic absorption of calcium ox- alate in water solvent shows close agreement with the experimental finding [36] compared to the re- sults obtained in the gas phase [14]. The majority of charge density is concentrated around the carbon and oxygen atoms in HOMO, LUMO, and HOMO-1 orbitals. However, in LUMO+1, the charge density localized around calcium atom (Figure 4b). This vi- sual representation reflects the charge distribution within the compound [37]. The energy gap, differ- ence between the energies of HOMO and LUMO orbitals, is found to be 5.90 eV (Figure 4a). The number of states per unit energy interval at a given energy level in both occupied and virtual orbitals is illustrated with the DOS spectrum in Figure 5. The HOMO and LUMO orbitals and the spectra of DOS resemble closely each other. These results suggest that calcium oxalate can promote charge transfer process and exhibits enhanced capability for biological activities [38]. Figure 4: Calculated electronic absorption of cal- cium oxalate: UV-Vis spectrum (a) and the frontier molecular orbitals significantly contributing to the electronic transitions (b). Figure 5: Density of state spectrum along with the occupied and virtual orbitals in calcium oxalate. Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 315 Table 1: Calculated electronic properties of calcium oxalate. Maximum absorption wavelength (nm) Oscillatory strength Major contributions Absorption wavelength of whewellite (nm) [36] 278 0.000 HOMO → LUMO (99%) 288 265 0.000 HOMO-1 → LUMO (99%) - 231 0.008 HOMO → LUMO+1 (97%) 236 Table 2: Calculated global reactivity descriptors of calcium oxalate. Ionization potential (I) (eV) Electron affinity (A) (eV) Chemical hardness (η) (eV) Chemical softness (S) ( eV)−1 Electronic chemical potential (µ) (eV) Global electrophilicity index (ω) (eV) 6.48 0.58 2.95 0.34 -3.53 2.11 3.2.3 Global reactivity descriptors The chemical hardness and electrophilicity index of calcium oxalate is obtained to be 2.95 eV and 2.11 eV, respectively (Table 2). These positive values of chemical hardness and electrophilicity index sug- gest that the compound is suitable for charge trans- fer processes and can influence the binding energy in protein-ligand interactions [39]. 3.2.4 Molecular electrostatic potential and Mulliken atomic charges Polar regions around the atoms of calcium oxalate are demonstrated with different color codes rang- ing from -0.316 a.u. to 0.316 a.u. using MEP map. The intense positive potential is observed around the Ca7 and negative potential is found around the O1, O2, O3, and O4 atoms (Figure 6a). Figure 6: MEP map with contour lines (a) and Mulliken atomic charges contributed by each atom (b) in calcium oxalate molecule. Further, calcium atom has the maximum pos- itive Mulliken atomic charges and O1 as well as O4 show the maximum negative Mulliken atomic charges (Figure 6b). The variation of charges in oxygen atoms illustrates the induction effect of cal- cium atom to the bonded oxygen atoms. The MEP map and Mulliken atomic charge distribution show the similar nucleophilic and electrophilic regions in calcium oxalate. The reactive nature of the chem- ical compound associated with the polar property can be explained using MEP and Mulliken atomic charges [40]. And, the polar nature of calcium ox- alate is significant for the bonded and non-bonded interactions with lysozyme protein (Figure 9). 3.3 Interactions of CaC2O4 with lysozyme Lysozyme protein (7xf6.pdb) contains an acetate ion (C2H3O2) as a native ligand, which forms non bonded interactions with Ile77, Trp82, Ala126, and Trp127 and hydrogen-bonded (H-bonded) in- teraction with Asn78 (Figure 8a). All non-glycine residues except Cys134 and Ser54 confine within the most allowed regions of the Ramachandran plot Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 316 (Figure 7 and Table 3), indicating that the structure of lysozyme is valid and suitable for the protein- ligand interactions [41,42]. Figure 7: Ramachandran plots: all residues of lysozyme (a) and residues interacting with calcium oxalate (b). Table 3: Dihedral angle pairs (in degree) of active amino acids of lysozyme interacting with calcium oxalate. Active amino acids of lysozyme protein Dihedral angle (ϕ) Dihedral angle (ψ) Ile77 -75.37 134.83 Asn78 -92.15 142.91 Trp82 -110.81 -35.76 Val117 -52.71 -49.32 Ala126 -57.33 -30.91 Trp127 -93.36 117.50 The binding pocket identified using CASTp (Figure 8b) includes active residues Glu53, Asp71, Gln76, Ile77, Asn78, Trp82, Val117, Ala126, Trp127, Val128, and Ala129 with a total binding pocket area 71.823 Å2 and volume 49.887 Å3, re- spectively. This result suggests that the region identified by CASTp closely matches with the loca- tion of native ligand, making it suitable for molec- ular docking between lysozyme and calcium ox- alate. The visualizations of the best docked pose of lysozyme-calcium oxalate complex within the bind- ing pocket are shown in Figure 9. The residue Asn78 forms H-bond, while the residues Ile77, Trp82, Val117, Ala126, and Trp127 contribute in hydrophobic interactions with calcium oxalate ( Figures 9b,c). These interactions contribute the binding energy of −4.18 kcal/mol (Table 4). This result indicates that the binding affinity of calcium oxalate with lysozyme is slightly higher than that of lactoferrin as shown in our previous work [14]. The present molecular docking analysis suggests that the calcium oxalate, a key component in kidney stones, exhibits a high binding efficacy within the active region of the lysozyme, promoter protein in neprolithiasis. Lysozyme is one of the key proteins found in the matrix of whewellite-type urinary cal- culi [16]. In in vivo conditions, matrix proteins of the calculi can exhibit aggregation-inducing proper- ties, which can enhance the particle size of calcium oxalate type crystals, facilitating crystal growth in urinary tract [43]. The location, size, and type of urinary calculi in a urinary tract significantly influence the treatment modalities. Calculi larger than 7 mm are unlikely to Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 317 move spontaneously through urine and typically re- quire surgical intervention. Further, calculi ranging from 1.1 cm to 2.4 cm in size are ideal for break- ing using the extracorporeal shockwave lithotripsy (ESWL) and greater than 2 cm are recommended for percutaneous nephrolithotomy (PCNL) tech- niques [44–46]. Among different calculi, weddel- lite types are relatively easy to break. In con- trast, whewellite, infected struvite, and cystine of- fer the highest resistance to the ESWL. Generally, Ureteroscopy (URS) is better option for treatment of cystine type calculi [44, 46]. Figure 8: Interactions of native ligand, acetate ion (C2H3O2), with lysozyme (7xf6.pdb) are represented by spoked arcs for nonbonded residues, green-dotted line for hydrogen bond, and written in green color for hydrogen-bonded residue using LigPlot+ v.2.2 (a). The location of the binding pocket highlighted in red within the lysozyme represented using ribbon like structure generated by CASTp (b). Figure 9: Calcium oxalate (red) within the binding pocket of lysozyme (green for interacting and blue for non interacting residues) visualized using PyMOL (a,b). Interactions of calcium oxalate with lysozyme are illustrated with nonbonded residues shown as spoked arcs, hydrogen-bonded residue indicated in green color, generated using LigPlot+ v.2.2 (c). Table 4: Molecular docking results of calcium oxalate with lysozyme at temperature 298.15 K. Energy components Energy (kcal/mol) van der Waals, hydrogen bond, and disolvation energy -4.16 Electrostatic Energy -0.02 Total binding energy -4.18 Arjun Acharya et al./ BIBECHANA 21 (2024) 311-320 318 4 Conclusion This work is focused on the characterization of kid- ney stones and the study of nephrolithiasis phenom- ena using FTIR, DFT, and molecular docking ap- proaches. The characterization of kidney stones us- ing spectroscopic techniques reveals that the major- ity are composed of calcium oxalate monohydrate. The quantum mechanical properties suggest that calcium oxalate can interact through bonded and non-bonded interactions with proteins. The molec- ular docking analysis of calcium oxalate within the binding regions of lysozyme shows effective binding interactions. Hence, lysozyme can play potential role for the development of calcium oxalate type nephrolithiasis. In summary, this research finds the distribution of the types of kidney stones and con- tributes to understand the phenomena of calcium oxalate type nephrolithiasis, which remains still un- clear and is one of the most challenging global prob- lems for the scientific community. Further, charac- terization of kidney stones from different regions us- ing a large sample size and the study of nephrolithi- asis using different promoter and inhibitor macro- molecules from both experimental and in silico ap- proaches are still necessary for a detailed under- standing of this global challenge. Competing interests The authors declare that there is no conflict of in- terest. Ethics approval and consent to participate This study was conducted with the approval of the institutional review committee of the Institute of Medicine, Tribhuvan University Teaching Hospital, Maharajgunj, Kathmandu, Nepal with an approval number of 117 (6-11) E2 079/080. Informed con- sent was received from participants or their parents or legal guardians. Data availability The data supporting the findings of this study are available within the article. Authors’ contributions A Acharya: Conceived and designed the experi- ments, performed data analysis, prepared the fig- ures, and wrote the manuscript M Khanal: Data analysis, manuscript writing R Maharjan: Technical support, critical feed back and revised the manuscript K Gyawali: Critical feed back and revised the manuscript K Khanal: Critical feed back and revised the manuscript MB Kshetri: Critical feed back and revised the manuscript BR Luitel: Critical feedback, data analysis, and re- vised the manuscript R Adhikari: Critical feedback, data analysis, and revised the manuscript DD Mulmi: Critical feedback and revised the manuscript TR Lamichhane: Technical support, critical feed- back, data analysis, and revised the manuscript HP Lamichhane: Critical feedback, data analysis and revised the manuscript Acknowledgment We would like to express our gratitude to Prof. Dr. Rajendra Parajuli and Asst. Prof. Pitamber Shrestha, Amrit campus, T. 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