Decrease of catalytic efficiency of Photinus pyralis firefly luciferase in the presence of graphene quantum dots

Document Type : Research Paper


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Medical Biotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran

3 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran


Objective(s): Firefly luciferase is a monooxygenase enzyme that emits flash of light during the enzymatic reaction. Luciferase has been used in many bioanalytical fields from ATP detection methods to in vivo imaging. In recent decades, focus has been carried out on nanoparticles for their fluorescence properties. Semiconductor quantum dots have unique tunable properties that turn them promising tools in biological and biomedical researches, as nanosensors, photo-electrochemical and light-emitting devices. Carbon-based nanoparticles such as graphene quantum dots (GQDs) have useful benefits such as low toxicity, suitable luminescence and easy preparation.
Materials and Methods: In this study, recombinant P. pyralis luciferase was expressed and purified based on N-terminal His-tag and then kinetic parameters of enzyme activity such as Km and Vmax values in presence and absence of GQDs were calculated.
Results: The results showed that Km for ATP and luciferin substrates in the presence of GQDs were increased. Fluorescence spectroscopy showed significant changes in protein structure or in fluorescence spectra and decrease in the activity of the luciferase in presence of GQD. Both loss of activity and increase of substrates Km showed decrease of catalytic efficiency presumably through structural alteration.
Conclusion: From these data it can be concluded that the protein structure under the influence of GQD may have changed that lead to alteration of enzyme activity.


1.Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, K Hiroaki. Structural basis for the spectral difference in luciferase bioluminescence. Nature. 2006; 440(7082): 372-376.
2. Ebrahimi M, Hosseinkhani S, Heydari A, Khavari-Nejad RA, Akbari J. Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator. Appl Biochem Biotech. 2012; 168(3): 604-615.
3.Kargar F, Mortazavi M, Savardashtaki A, Hosseinkhani S, Mahani MT, Ghasemi Y. Genomic and protein structure analysis of the luciferase from the Iranian bioluminescent beetle, Luciola sp. Int J Biol Macromol. 2019; 124: 689-698.
4. Zomorodimanesh S, Hosseinkhani S, Baharifar H, Yousefi F, Farsad J. Expression and Purification of Firefly Luciferase and its Interaction with Cadmium Telluride Quantum Dot. Bmmj. 2019; 5(1): 35-46.
5. Ataei F, Hosseinkhani S, Khajeh K. Limited proteolysis of luciferase as a reporter in nanosystem biology: a comparative study. Photochem Photobiol. 2009; 85(5): 1162-1167.
6. Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996; 4(3): 287-298.
7. De Wet JR, Wood KV, Helinski DR, DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. P Natl Acad Sci. 1985; 82(23): 7870-7873.
8. Gould SJ, Subramani S. Firefly luciferase as a tool in molecular and cell biology. Anal Biochem. 1988; 175(1): 5-13.
9. Ebrahimi M, Hosseinkhani S, Heydari A, Khavari-Nejad RA, Akbari J. Controversial effect of two methylguanidine-based ionic liquids on firefly luciferase. Photoch Photobio Sci. 2012; 11(5): 828-834.
10. Branchini BR, Southworth TL, Khattak NF, Michelini E, Roda A. Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem. 2005; 345(1): 140-148.
11. Tisi L, White P, Squirrell D, Murphy M, Lowe C, Murray J. Development of a thermostable firefly luciferase. Anal Chim Acta. 2002; 457(1): 115-123.
12. Marques SM, Esteves da Silva JC. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions. Iubmb. 2009; 61(1): 6-17.
13. Fraga H. Firefly luminescence: a historical perspective and recent developments. PHOTOCH PHOTOBIO SCI. 2008; 7(2): 146-158.
14. Moradi M, Hosseinkhani S, Emamzadeh R. Implication of an unfavorable residue (Thr346) in intrinsic flexibility of firefly luciferase. Enzyme Microb Tech. 2012; 51(4): 186-192.
15. Fraga H, Fernandes D, Novotny J, Fontes R, Esteves da Silva JC. Firefly luciferase produces hydrogen peroxide as a coproduct in dehydroluciferyl adenylate formation. Chembiochem. 2006; 7(6): 929-935.
16. Baldwin TO. Firefly luciferase: the structure is known, but the mystery remains. Structure. 1996; 4(3): 223-228.
17. Noori AR, Hosseinkhani S, Ghiasi P, Akbari J, Heydari A. Magnetic nanoparticles supported ionic liquids improve firefly luciferase properties. Appl Biochem Biotech. 2014; 172(6): 3116-27.
18. Lohrasbi-Nejad A, Torkzadeh-Mahani M, Hosseinkhani S. Hydrophobin-1 promotes thermostability of firefly luciferase. Febs J. 2016; 283(13): 2494-2507
19. Noori AR, Hosseinkhani S, Ghiasi P, Heydari A, Akbari J. Water-miscible ionic liquids as novel effectors for the firefly luciferase reaction. Eng Life Sci. 2013; 13(2): 201-209.
20. Sharma A, Gupata MK, Gupta R. Quantum phenomena in zero dimensions: quantum dots. Ijritcc. 2013; 1(1): 1-18
21. Peng C-W, Li Y. Application of Quantum Dots-Based Biotechnology in Cancer Diagnosis: Current Status and Future Perspectives. Journal of Nanomaterials. 2010[(article ID 676839): 11 pp]
22. Mansur A, Mansur H, González J. Enzyme-polymers conjugated to quantum-dots for sensing applications. Sensors. 2011; 11(10): 9951-9972.
23. Byers RJ, Hitchman ER. Quantum dots brighten biological imaging. Prog Histochem Cyto. 2011; 45(4): 201-237.
24. Gill R, Zayats M, Willner I. Semiconductor quantum dots for bioanalysis.  Angew Chem Int Edit. 2008; 47(40): 7602-7625.
25. Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, Southworth TL. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem. 2007; 361(2): 253-262.
26. Tang L, Ji R, Cao X, Lin J, Jiang H, Li X, Teng Ks, Luk Cm, Zeng S, Hao J, Lau Sp. Deep ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots.  Acs Nano. 2012; 6(6): 5102-5110.
27. Chen W, Lv G, Hu W, Li D, Chen S, Dai Z. Synthesis and applications of graphene quantum dots: a review. Nanotechnology Reviews. 2018; 7(2): 157-185.
28. Xiaoyan Z, Hongxia B, Zaijun L, Junkang L. Graphene quantum dot-modified lipase for synthesis of L-menthyl acetate with improved activity, stability and thermostability. Adv Synth Catal. 2016; 1: 1-6.
29. Pinto-Alphandary H, Andremont A, Couvreur P. Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. Int J Antimicrob Ag. 2000; 13(3): 155-168.
30. Pumera M, Ambrosi A, Bonanni A, Chng ELK, Poh HL. Graphene for electrochemical sensing and biosensing. Trac-Trend Anal Chem. 2010; 29(9): 954-965.
31. Shehab M, Ebrahim S, Soliman M. Graphene quantum dots prepared from glucose as optical sensor for glucose.  J Lumin. 2017; 184: 110-116.
32. Pan D, Zhang J, Li Z, Wu M. Hydrothermal route for cutting graphene sheets into blue-luminescent graphene quantum dots. Adv Mater. 2010; 22(6): 734-738.
33. Moases Ghafary S, Nikkhah M, Hatamie S, Hosseinkhani S. Simultaneous Gene Delivery and Tracking through Preparation of Photo-Luminescent Nanoparticles Based on Graphene Quantum Dots and Chimeric Peptides. Sci Rep. 2017; 7: 9552.
34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72(1-2): 248-254.
35. Mir M, Ishtiaq S, Rabia S, Khatoon M, Zeb A, Khan GM, Rehmen Au, Din Fu. Nanotechnology: from in vivo imaging system to controlled drug delivery. Nanoscale Res Lett. 2017; 12(1): 500.
36. Bilal M, Mehmood S, Rasheed T, Iqbal H. Bio-catalysis and biomedical perspectives of magnetic nanoparticles as versatile carriers. Magncz. 2019; 5(3): 42.
37. Mehrabi M, Hosseinkhani S, Ghobadi S. Stabilization of firefly luciferase against thermal stress by osmolytes. Int J Biol Macromol. 2008; 43(2): 187-191.
38. Käkinen A, Ding F, Chen P, Mortimer M, Kahru A, Ke PC. Interaction of firefly luciferase and silver nanoparticles and its impact on enzyme activity. NANOTECHNOL. 2013; 24(34): 345101.
39. Gupta S, Smith T, Banaszak A, Boeckl J. Graphene Quantum Dots Electrochemistry and Development of Ultrasensitive Enzymatic Glucose Sensor. MRS Advances. 2018; 3(15-16): 831-847.
40. Muthurasu A, Ganesh V. Horseradish peroxidase enzyme immobilized graphene quantum dots as electrochemical biosensors. Appl Biochem Biotech. 2014; 174(3): 945-959.
41. Hosseinkhani S. Molecular enigma of multicolor bioluminescence of firefly luciferase. Cell Mol Life Sci. 2011; 68(7): 1167-1182.
42. Alipour BS, Hosseinkhani S, Ardestani SK, Moradi A. The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase. Photoch Photobio Sci. 2009; 8(6): 847-55.
43. Amini-Bayat Z, Hosseinkhani S, Jafari R, Khajeh K. Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase.  Bba-Proteins Proteom. 2012; 1824(2): 350-358.