In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)‚ was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study.
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Wang Y, Wang L, Zhu M, Xue J, Hua R, Li QX. Comparative studies on biophysical interactions between gambogic acid and serum albumin via multispectroscopic approaches and molecular docking. J Lumin. 2019;205:210–8.
Wang Y, Liu J, Zhu M, Wang L, Zen X, Fan S, et al. Biophysical characterization of interactions between falcarinol-type polyacetylenes and human serum albumin via multispectroscopy and molecular docking techniques. J Lumin. 2018;200:111–9.
Wang Y, Zhu M, Liu F, Wu X, Pan D, Liu J, et al. Comparative studies of interactions between fluorodihydroquinazolin derivatives and human serum albumin with fluorescence spectroscopy. Molecules. 2016;21(10):1373–86.
Swain BC, Subadini S, Rout J, Sakshi MPP, Sahoo H, Tripathy U. Biophysical study on complex formation between β-Lactoglobulin and vitamin B12. Food Chem. 2020;312:126064.
Zaman M, Safdari HA, Khan AN, Zakariya SM, Nusrat S, Chandel TI, et al. Interaction of anticancer drug pinostrobin with lysozyme: a biophysical and molecular docking approach. J Biomol Struct Dyn. 2019;37(16):4338–44.
Millan S, Satish L, Bera K, Susrisweta B, Singh DV, Sahoo H. A spectroscopic and molecular simulation approach toward the binding affinity between lysozyme and phenazinium dyes: an effect on protein conformation. J Phys Chem B. 2017;121(7):1475–84.
Pawar SK, Naik RS, Seetharamappa J. Exploring the binding of two potent anticancer drugs bosutinib and imatinib mesylate with bovine serum albumin: spectroscopic and molecular dynamic simulation studies. Anal Bioanal Chem. 2017;409(27):6325–35.
Fleming A, Allison VD. Observations on a bacteriolytic substance (“lysozyme”) found in secretions and tissues. Br J Exp Pathol. 1922;3(5):252–60.
Teichberg VI, Sharon N. A spectrofluorometric study of tryptophan 108 in hen egg-white lysozyme. FEBS Lett. 1970;7(2):171–4.
Sheng C, Dian HJSS. Press T. Lysozyme. 1982:50–1.
Imoto T, Forster LS, Rupley JA, Tanaka F. Fluorescence of lysozyme: emissions from tryptophan residues 62 and 108 and energy migration. Proc Natl Acad Sci U S A. 1972;69(5):1151.
Jash C, Payghan PV, Ghoshal N, Suresh KG. Binding of the iminium and alkanolamine forms of sanguinarine to lysozyme: spectroscopic analysis, thermodynamics, and molecular modeling studies. J Phys Chem B. 2014;118(46):13077–91.
Strynadka NCJ, James MNG. Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D. J Mol Biol. 1991;220(2):401–24.
Primo ED, Otero LH, Ruiz F, Klinke S, Giordano W. The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook. Biochem Mol Biol Educ. 2018;46(1):83–90.
Panja S, Halder M. Exploration of electrostatic interaction in the hydrophobic pocket of lysozyme: importance of ligand-induced perturbation of the secondary structure on the mode of binding of exogenous ligand and possible consequences. J Photochem Photobiol B. 2016;161:253–65.
Sava G, Benetti A, Ceschia V, Pacor S. Lysozyme and cancer: role of exogenous lysozyme as anticancer agent (review). Anticancer Res. 1989;9(3):583–91.
Khan MI, Dowarha D, Katte R, Chou R-H, Filipek A, Yu C. Lysozyme as the anti-proliferative agent to block the interaction between S100A6 and the RAGE V domain. PLoS One. 2019;14(5):e0216427.
Patel TV, Rennke HG, Sloan JM, DeAngelo DJ, Charytan DM. A forgotten cause of kidney injury in chronic myelomonocytic leukemia. Am J Kidney Dis. 2009;54(1):159–64.
Santoriello D, Andal LM, Cox R, D’Agati VD, Markowitz GS. Lysozyme-induced nephropathy. Kidney Int Rep. 2017;2(1):84–8.
Das S, Rohman MA, Singha RA. Exploring the non-covalent binding behaviours of 7-hydroxyflavone and 3-hydroxyflavone with hen egg white lysozyme: multi-spectroscopic and molecular docking perspectives. J Photochem Photobiol B. 2018;180:25–38.
Cao D, Wu H, Li Q, Sun Y, Liu T, Fei J, et al. Expression of recombinant human lysozyme in egg whites of transgenic hens. PLoS One. 2015;10(2):e0118626.
Blake C, Swan I. X-ray analysis of structure of human lysozyme at 6 Å resolution. Nat New Biol. 1971;232(27):12–5.
Philips FS, Sternberg SS, Hamilton L, Clarke DA. The toxic effects of 6-mercaptopurine and related compounds. Ann N Y Acad Sci. 1954;60(2):283–96.
Bostrom B, Erdmann G. Cellular pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia. Am J Pediatr Hematol Oncol. 1993;15(1):80–6.
Adler DJ, Korelitz BI. The therapeutic efficacy of 6-mercaptopurine in refractory ulcerative colitis. Am J Gastroenterol. 1990;85(6).
Timmer A, Patton PH, Chande N, McDonald JWD, MacDonald JK. Azathioprine and 6-mercaptopurine for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2016;5.
O'Brien JJ, Bayless TM, Bayless JA. Use of azathioprine or 6-mercaptopurine in the treatment of Crohn’s disease. Gastroenterology. 1991;101(1):39–46.
Nielsen OH, Vainer B, Rask-Madsen J. The treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprine. Aliment Pharmacol Ther. 2001;15(11):1699–708.
Lennard L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol. 1992;43(4):329–39.
Pabbathi A, Ghosh S, Samanta A. FCS study of the structural stability of lysozyme in the presence of morpholinium salts. J Phys Chem B. 2013;117(51):16587–93.
Tang D, Liao D, Zhu Q, Wang F, Jiao H, Zhang Y, et al. Fluorescence turn-on detection of a protein through the displaced single-stranded DNA binding protein binding to a molecular beacon. Chem Commun. 2011;47(19):5485–7.
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30(16):2785–91.
Mukherjee SK, Mandal RS, Das S, Mukherjee M. Effect of non-β-lactams on stable variants of inhibitor-resistant TEM β-lactamase in uropathogenic Escherichia coli: implication for alternative therapy. J Appl Microbiol. 2018;124(3):667–81.
Release S. 3: Desmond molecular dynamics system, DE Shaw research, New York, NY, 2017. Maestro-Desmond interoperability tools, Schrödinger, New York, NY. 2017.
Abdul Samad F, Suliman BA, Basha SH, Manivasagam T, Essa MM. A comprehensive in silico analysis on the structural and functional impact of SNPs in the congenital heart defects associated with NKX2-5 gene—a molecular dynamic simulation approach. PLoS One. 2016;11(5):e0153999.
Studio DJSD, CA : http://www.accelrys. com. 2.5 Software Package AI.
Jing M, Song W, Liu R. Binding of copper to lysozyme: spectroscopic, isothermal titration calorimetry and molecular docking studies. Spectrochim Acta A Mol Biomol Spectrosc. 2016;164:103–9.
Lakowicz JR. Principles of fluorescence spectroscopy: Springer Science & Business Media; 2013.
Katrahalli U, Jaldappagari S, Kalanur SS. Study of the interaction between fluoxetine hydrochloride and bovine serum albumin in the imitated physiological conditions by multi-spectroscopic methods. J Lumin. 2010;130(2):211–6.
Mitra P, Pal U, Chandra Maiti N, Ghosh A, Bhunia A, Basu S. Identification of modes of interactions between 9-aminoacridine hydrochloride hydrate and serum proteins by low and high resolution spectroscopy and molecular modeling. RSC Adv. 2016;6(58):53454–68.
Wei XL, Xiao JB, Wang Y, Bai Y. Which model based on fluorescence quenching is suitable to study the interaction between trans-resveratrol and BSA? Spectrochim Acta A Mol Biomol Spectrosc. 2010;75(1):299–304.
Paul BK, Ghosh N, Mukherjee S. Binding interaction of a prospective chemotherapeutic antibacterial drug with β-lactoglobulin: results and challenges. Langmuir. 2014;30(20):5921–9.
Wang J, Yang X, Wang J, Xu C, Zhang W, Liu R, et al. Probing the binding interaction between cadmium(ii) chloride and lysozyme. New J Chem. 2016;40(4):3738–46.
Rudra S, Jana A, Sepay N, Patel BK, Mahapatra A. Characterization of the binding of strychnine with bovine β-lactoglobulin and human lysozyme using spectroscopic, kinetic and molecular docking analysis. New J Chem. 2018;42(11):8615–28.
Sun Z, Liu Y, Li Y. Selective recognition of 6-mercaptopurine based on luminescent metal–organic frameworks Fe-MIL-88NH2. Spectrochim Acta A Mol Biomol Spectrosc. 2015;139:296–301.
Nad S, Pal H. Electron transfer from aromatic amines to excited coumarin dyes: fluorescence quenching and picosecond transient absorption studies. J Phy Chem A. 2000;104(3):673–80.
Swain BC, Mishra PP, Mishra H, Tripathy U. Monitoring the binding of serotonin to silver nanoparticles: a fluorescence spectroscopic investigation. J Photoch Photobio A. 2018;367:219–25.
Patra D, Barakat C, Tafech RM. Study on effect of lipophilic curcumin on sub-domain IIA site of human serum albumin during unfolded and refolded states: a synchronous fluorescence spectroscopic study. Colloids Surf B: Biointerfaces. 2012;94:354–61.
Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006;1(6):2876–90.
Millan S, Satish L, Kesh S, Chaudhary YS, Sahoo H. Interaction of lysozyme with rhodamine B: a combined analysis of spectroscopic & molecular docking. J Photochem Photobiol B. 2016;162:248–57.
Chen Y-H, Yang JT, Martinez HM. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry. 1972;11(22):4120–31.
Das A, Thakur R, Dagar A, Chakraborty A. A spectroscopic investigation and molecular docking study on the interaction of hen egg white lysozyme with liposomes of saturated and unsaturated phosphocholines probed by an anticancer drug ellipticine. Phys Chem Chem Phys. 2014;16(11):5368–81.
Basak S, Prasad GVRK, Varkey J, Chattopadhyay K. Early sodium dodecyl sulfate induced collapse of α-synuclein correlates with its amyloid formation. ACS Chem Neurosci. 2015;6(2):239–46.
Maity BK, Das AK, Dey S, Moorthi UK, Kaur A, Dey A, et al. Ordered and disordered segments of amyloid-β drive sequential steps of the toxic pathway. ACS Chem Neurosci. 2019;10(5):2498–509.
Kohl T, Schwille P. Fluorescence correlation spectroscopy with autofluorescent proteins. In: Rietdorf J, editor. Microscopy techniques: -/- Berlin, Heidelberg: Springer Berlin Heidelberg; 2005. p. 107–42.
Sen Mojumdar S, Chowdhury R, Chattoraj S, Bhattacharyya K. Role of ionic liquid on the conformational dynamics in the native, molten globule, and unfolded states of cytochrome C: a fluorescence correlation spectroscopy study. J Phys Chem B. 2012;116(40):12189–98.
Sherman E, Itkin A, Kuttner YY, Rhoades E, Amir D, Haas E, et al. Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins. Biophys J. 2008;94(12):4819–27.
The authors thank Mr. Ajaya Das, Chemical Sciences Division, Saha Institute of Nuclear Physics (SINP), Kolkata, India for his assistance in time-resolved fluorescence measurements and preliminary data analysis. We also thank Dr. Anand Kant Das and Dr. Shrutidhara Biswas for the helpful scientific discussion.
The authors acknowledge the Indian Institute of Technology (Indian School of Mines), Dhanbad, India for infrastructure facility and financial assistance (FRS (74)/2014-15/APH). For the computational facility, we are thankful to the Ministry of Human Resource Development (MHRD), New Delhi, India for financial assistance (MHRD/(FDC)/2015-2016/438/INST).
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Swain, B.C., Mukherjee, S.K., Rout, J. et al. A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine. Anal Bioanal Chem (2020). https://doi.org/10.1007/s00216-020-02483-1
- Fluorescence spectroscopy
- Circular dichroism
- Fluorescence correlation spectroscopy
- MD simulation