Interactions and effects of food additive dye Allura red on pepsin structure and protease activity; experimental and computational supports

Fatemeh Balaei , Mohabbat Ansari, Negin Farhadian, Sajad Moradi , Mohsen Shahlaei


Background and purpose: Today, color additives such as Allura red (AR) are widely used in different kinds of food products. Pepsin is a globular protein that is secreted as a digestive protease from the main cells in the stomach. Because of the important role of pepsin in protein digestion and because of its importance in digestive diseases the study of the interactions of pepsin with chemical food additives is important.

Experimental approach: In this study, the interactions between AR and pepsin were investigated by different computational and experimental approaches such as ultraviolet and fluorescence spectroscopy along with computational molecular modeling.

Findings/Results: The experimental results of fluorescence indicated that AR can strongly quench the fluorescence of pepsin through a static quenching. Thermodynamic analysis of the binding phenomena suggests that van der Waals forces and hydrogen bonding played a major role in the complex formation. The results of synchronous fluorescence spectra and furrier transformed infra-red (FTIR) experiments showed that there are no significant structural changes in the protein conformation. Also, examined pepsin protease activity revealed that the activity of pepsin was increased upon ligand binding. In agreement with the experimental results, the computational results showed that hydrogen bonding and van der Waals interactions occurred between AR and binding sites.

Conclusion and implications: From the pharmaceutical point of view, this interaction can help us to get a deeper understanding of the effect of this synthetic dye on food digestion.



Allura red; Enzyme activity; Molecular dynamics simulation; Pepsin; Spectroscopy study.

Full Text:



Oplatowska-Stachowiak M, Elliott CT. Food colors: existing and emerging food safety concerns. Crit Rev Food Sci Nutr. 2017;57(3):524-548.

DOI: 10.1080/10408398.2014.889652.

Downham A, Collins P. Colouring our foods in the last and next millennium. Int J Food Sci Technol. 2000;35(1):5-22.

DOI: 10.1046/j.1365-2621.2000.00373.x.

Stevens LJ, Burgess JR, Stochelski MA, Kuczek T. Amounts of artificial food dyes and added sugars in foods and sweets commonly consumed by children. Clin Pediatr (Phila). 2015;54(4):309-321.

DOI: 10.1177/0009922814530803.

Wang L, Zhang G, Wang Y. Binding properties of food colorant allura red with human serum albumin in vitro. Mol Biol Rep. 2014;41(5):3381-3391.

DOI: 10.1007/s11033-014-3200-z.

Ying M, Huang F, Ye H, Xu H, Shen L, Huan T, et al. Study on interaction between curcumin and pepsin by spectroscopic and docking methods. Int J Biol Macromol. 2015;79:201-208.

DOI: 10.1016/j.ijbiomac.2015.04.057.

Shen L, Xu H, Huang F, Li Y, Xiao H, Yang Z, et al. Investigation on interaction between Ligupurpuroside A and pepsin by spectroscopic and docking methods. Spectrochim Acta A Mol Biomol Spectrosc. 2015;135:256-263.

DOI: 10.1016/j.saa.2014.06.087.

Bijari N, Balalaie S, Akbari V, Golmohammadi F, Moradi S, Adibi H, et al. Effective suppression of the modified PHF6 peptide/1N4R Tau amyloid aggregation by intact curcumin, not its degradation products: another evidence for the pigment as preventive/therapeutic “functional food”. Int J Biol Macromol. 2018;120(Pt A):1009-1022.

DOI: 10.1016/j.ijbiomac.2018.08.175.

Jafari F, Moradi S, Nowroozi A, Sadrjavadi K, Hosseinzadeh L, Shahlaei M. Exploring the binding mechanism of paraquat to DNA by a combination of spectroscopic, cellular uptake, molecular docking and molecular dynamics simulation methods. New J Chem. 2017;41:14188-14198.

DOI: 10.1039/C7NJ01645J.

Moradi S, Hosseini E, Abdoli M, Khani S, Shahlaei M. Comparative molecular dynamic simulation study on the use of chitosan for temperature stabilization of interferon αII. Carbohydr Polym. 2019;203:52-59.

DOI: 10.1016/j.carbpol.2018.09.032.

Moradi S, Khani S, Ansari M, Shahlaei M. Atomistic details on the mechanism of organophosphates resistance in insects: insights from homology modeling, docking and molecular dynamic simulation. J Mol Liq. 2019;276:59-66.

DOI: 10.1016/j.molliq.2018.11.152.

Wu D, Yan J, Wang J, Wang Q, Li H. Characterisation of interaction between food colourant allura red AC and human serum albumin: multispectroscopic analyses and docking simulations. Food Chem. 2015;170:423-429.

DOI: 10.1016/j.foodchem.2014.08.088.

Zhao L, Guo R, Sun Q, Lan J, Li H. Interaction between azo dye Acid Red 14 and pepsin by multispectral methods and docking studies. Luminescence. 2017;32(7):1123-1130.

DOI: 10.1002/bio.3298.

Moradi S, Farhadian N, Balaei F, Ansari M, Shahlaei M. Multi spectroscopy and molecular modeling aspects related to drug interaction of aspirin and warfarin with pepsin; structural change and protease activity. Spectrochim Acta A Mol Biomol Spectrosc. 2020;228:117813,1-29.

DOI: 10.1016/j.saa.2019.117813.

Krejpcio Z, Wojciak R. The influence of Al3+ ions on pepsin and trypsin activity in vitro. Pol J Environ Stud. 2002;11(3):251-254.

Morris GM, Huey R, Olson AJ. Using autodock for ligand‐receptor docking. Curr Protoc Bioinformatics. 2008;24(1):8.14.1-8.14.40.

DOI: 10.1002/0471250953.bi0814s24.

Morshedi D, Kesejini TS, Aliakbari F, Karami-Osboo R, Shakibaei M, Marvian AT, et al. Identification and characterization of a compound from Cuminum cyminum essential oil with antifibrilation and cytotoxic effect. Res Pharm Sci. 2014;9(6):431-443.

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The protein data bank. Nucleic Acids Res. 2000;28(1):235-242.

DOI: 10.1093/nar/28.1.235.

Rose PW, Beran B, Bi C, Bluhm WF, Dimitropoulos D, Goodsell DS, et al. The RCSB protein data bank: redesigned web site and web services. Nucleic Acids Res. 2011;39(Database issue):D392-D401.

DOI: 10.1093/nar/gkq1021.

Jalali F, Dorraji PS, Mahdiuni H. Binding of the neuroleptic drug, gabapentin, to bovine serum albumin: insights from experimental and computational studies. J Lumin. 2014;148:347-352.

DOI: 10.1016/j.jlumin.2013.12.046.

Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform. 2012;4(1):17-33.

DOI: 10.1186/1758-2946-4-17.

Bijari N, Moradi S, Ghobadi S, Shahlaei M. Elucidating the interaction of letrozole with human serum albumin by combination of spectroscopic and molecular modeling techniques. Res Pharm Sci. 2018;13(4):304-315.

DOI: 10.4103/1735-5362.235157.

Malde AK, Zuo L, Breeze M, Stroet M, Poger D, Nair PC, et al. An automated force field topology builder (ATB) and repository: version 1.0. J Chem Theory Comput. 2011;7(12):4026-4037.

DOI: 10.1021/ct200196m.

Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem. 2005;26(16):1701-1718.

DOI: 10.1002/jcc.20291.

Banisharif-Dehkordi F, Mobini-Dehkordi M, Shakhsi-Niaei M, Mahnam K. Design and molecular dynamic simulation of a new double-epitope tolerogenic protein as a potential vaccine for multiple sclerosis disease. Res Pharm Sci. 2019;14(1):20-26.

DOI: 10.4103/1735-5362.251849.

Wang K, Hu F, Xu K, Cheng H, Jiang M, Feng R, et al. CASCADE_SCAN: mining signal transduction network from high-throughput data based on steepest descent method. BMC bioinformatics. 2011;12(1):164-178.

DOI: 10.1186/1471-2105-12-164.

Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126(1):014101,1-8.

DOI: 10.1063/1.2408420.

Hess B, Kutzner C, Van Der Spoel D, Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput. 2008;4(3):435-447.

DOI: 10.1021/ct700301q.

Vaiwala R, Jadhav S, Thaokar R. Electrostatic interactions in dissipative particle dynamics Ewald-like formalism, error analysis, and pressure computation. J Chem Phys. 2017;146(12):124904, 1-10.

DOI: 10.1063/1.4978809.

Raza S, Azam SS. AFD: an application for bi-molecular interaction using axial frequency distribution. J Mol Model. 2018;24(4):84-92.

DOI: 10.1007/s00894-018-3601-3.

Mojaddami A, Sakhteman A, Fereidoonnezhad M, Faghih Z, Najdian A, Khabnadideh S, et al. Binding mode of triazole derivatives as aromatase inhibitors based on docking, protein ligand interaction fingerprinting, and molecular dynamics simulation studies. Res Pharm Sci. 2017;12(1):21-30.

DOI: 10.4103/1735-5362.199043.

Yan J, Zhang G, Hu Y, Ma Y. Effect of luteolin on xanthine oxidase: inhibition kinetics and interaction mechanism merging with docking simulation. Food Chem. 2013;141(4):3766-3773.

DOI: 10.1016/j.foodchem.2013.06.092.

Ghalandari B, Divsalar A, Saboury AA, Haertlé T, Parivar K, Bazl R, et al. Spectroscopic and theoretical investigation of oxali-palladium interactions with β-lactoglobulin. Spectrochim Acta A Mol Biomol Spectrosc. 2014;118:1038-1046.

DOI: 10.1016/j.saa.2013.09.126.

Ahmadi F, Ebrahimi-Dishabi N, Mansouri K, Salimi F. Molecular aspect on the interaction of zinc-ofloxacin complex with deoxyribonucleic acid, proposed model for binding and cytotoxicity evaluation. Res Pharm Sci. 2014;9(5):367-383.

Hou HN, Qi ZD, Ouyang YW, Liao FL, Zhang Y, Liu Y. Studies on interaction between Vitamin B12 and human serum albumin. J Pharm Biomed Anal. 2008;47(1):134-139.

DOI: 10.1016/j.jpba.2007.12.029.

Ranjbar S, Ghobadi S, Khodarahmi R, Nemati H. Spectroscopic characterization of furosemide binding to human carbonic anhydrase II. Int J Biol Macromol. 2012;50(4):910-917.

DOI: 10.1016/j.ijbiomac.2012.02.005.

Lakowicz JR, Malicka J, Gryczynski I, Gryczynski Z, Geddes CD. Radiative decay engineering: the role of photonic mode density in biotechnology. J Phys D Appl Phys. 2003;36(14):R240-R249.

DOI: 10.1088/0022-3727/36/14/203.

Wu X, Liu J, Wang Q, Xue W, Yao X, Zhang Y, et al. Spectroscopic and molecular modeling evidence of clozapine binding to human serum albumin at subdomain IIA. Spectrochim Acta A Mol Biomol Spectrosc. 2011;79(5):1202-1209.

DOI: 10.1016/j.saa.2011.04.043.

Chaturvedi SK, Ahmad E, Khan JM, Alam P, Ishtikhar M, Khan RH. Elucidating the interaction of limonene with bovine serum albumin: a multi-technique approach. Mol Biosyst. 2015;11(1):307-316.

DOI: 10.1039/c4mb00548a.

Balaei F, Ghobadi S. Hydrochlorothiazide binding to human serum albumin induces some compactness in the molecular structure of the protein: a multi-spectroscopic and computational study. J Pharm Biomed Anal. 2019;162:1-8.

DOI: 10.1016/j.jpba.2018.09.009.

Gao W, Li N, Chen Y, Xu Y, Lin Y, Yin Y, et al. Study of interaction between syringin and human serum albumin by multi-spectroscopic method and atomic force microscopy. J Mol Struct. 2010; 983(1-3):133-140.

DOI: 10.1016/j.molstruc.2010.08.042.

Moradi N, Ashrafi-Kooshk MR, Ghobadi S, Shahlaei M, Khodarahmi R. Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: does esterase activity affect microenvironment of drug binding sites on the protein? J Lumin. 2015;160:351-361.

DOI: 10.1016/j.jlumin.2014.11.019.

Xu ZQ, Yang QQ, Lan JY, Zhang JQ, Peng W, Jin JC, et al. Interactions between carbon nanodots with human serum albumin and γ-globulins: the effects on the transportation function. J Hazard Mater. 2016;301:242-249.

DOI: 10.1016/j.jhazmat.2015.08.062.

Naik PN, Nandibewoor ST, Chimatadar SA. Non-covalent binding analysis of sulfamethoxazole to human serum albumin: fluorescence spectroscopy, UV-vis, FT-IR, voltammetric and molecular modeling. J Pharm Anal. 2015;5(3):143-152.

DOI: 10.1016/j.jpha.2015.01.003.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.