SEVTAP BAKIR, VEYSEL KENAN ÇELİK, İsmail SARI, SERPİL ERŞAN

Grafen Oksitin Ksantin Oksidaz Aktivitesi Üzerine İn Vitro Etkisinin İncelenmesi

Grafen karbon atomlarının altıgen bir kafes şeklinde düzenlenmesi ile oluşan iki boyutlu bir yapıdır ve biyolojik sistemlerin de dahil olduğu çeşitli uygulama alanlarına sahiptir. Bu çalışmada grafen oksitin (GO) pürin katabolizmasında rol alan ksantin oksidaz (KO) enzim aktivitesi üzerine in vitro etkisinin incelenmesi amaçlanmıştır. GO' nun KO aktivitesi üzerine etkisini incelemek amacıyla 0.50, 1.00, 5.00 ve 10.00 ppm olmak üzere 4 farklı derişimde GO çözeltisi KO tepkime ortamına ilave edilmiş ve 5 farklı substrat (hipoksantin) derişiminde aktivite değerleri spektrofotometrik olarak belirlenmiştir. Km ve Vmax değerleri saptanmıştır. KO' nun Vmax değeri 0.83 µM Ürat/mgProtein/dakika, Km değeri 138.95 µM olarak hesaplanmıştır. Tepkime ortamına 0.50, 1.00, 5.00, 10.00 ppm GO eklendiğinde KO enzimin sırasıyla %36.1, 72.3, 77.1 ve 84.3 oranında inhibe olduğu tespit edilmiştir. Sonuç olarak, GO derişimi arttıkça enzimin hem Km hem de Vmax değerlerinde dikkate değer bir azalma olduğu ve substrat derişimi artırsak da enzim inhibisyonun devam ettiği tespit edilmiştir. Bu da inhibisyon tipinin unkompetetif olduğunu göstermektedir.

Investigation of the In Vitro Effects of Graphene Oxide on the Xanthine Oxidase Activity

Graphene is a two-dimensional structure which consists of carbon atoms arranged in a hexagonal lattice and has various application fields including biological systems. In this study the in vitro effects of graphene oxide (GO) on the activity of xanthine oxidase (XO) enzyme which plays a role in purine catabolism was aimed to investigate. In order to examine the effect of GO on XO activity, 4 different concentrations GO solution including 0.50, 1.00, 5.00 and 10.00 ppm were added to the XO reaction medium and activity values were determined spectrophotometrically at 5 different substrate concentrations (hypoxanthine). Km ( Michaelis-Menten equation) and Vmax values were determined. Vmax amd Km ( Michaelis-Menten equation) values were calculated as 0.83 µM urate/mg protein/min and 138.95 µM, respectively. When 0.50, 1.00, 5.00, 10.00 ppm GO were added to the reaction medium, it was found that 36.1, 72.3, 77.1 and 84.3 percent of the XO enzyme activity was inhibited, respectively. In conclusion, a remarkable reduction was determined in both Vmax and Km values of the enzyme as the concentration of GO goes up and it was found that enzyme inhibition remain unchanged even if we increase the substrate concentration. It showed that the inhibition type is uncompetitive inhibition.

PDF

___

Qiu Y., Wang Z., Owens A. C., Kulaots I., Chen Y., Kane A. B., Hurt R. H. 2014. Antioxidant chemistry of graphene-based materials and its role in oxidation protection technology. Nanoscale, 6(20), 11744-11755.
Fang J., Yin H., Liao L., Qin H., Ueda F, Uemura K., Eguchi K., Bhrate G., Maeda H. 2016. Water soluble PEG-conjugate of xanthine oxidase inhibitor, PEG–AHPP micelles, as a novel therapeutic for ROS related inflammatory bowel diseases. Journal of Controlled Release, 223, 188-196.
Chang Y., Yang S.T., Liu J.H., Dong E., Wang Y., Cao A., Liu Y., Wang H. 2011. In vitro toxicity evaluation of graphene oxide on A549 cells. Toxicology letters, 200(3), 201-210.
Hu W., Peng C., Luo W., Lv M., Li X., Li D., Fan C. 2010. Graphene-based antibacterial paper. ACS nano, 4(7), 4317-4323.
Liao, K. H., Lin, Y. S., Macosko, C. W., Haynes, C. L. 2011. Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts. ACS applied materials & interfaces, 3(7), 2607-2615.
Wang, K., Ruan, J., Song, H., Zhang, J., Wo, Y., Guo, S., Cui, D. 2011. Biocompatibility of graphene oxide. Nanoscale Res Lett, 6(8), 1.
Cazorla, C., Rojas-Cervellera, V., Rovira, C. 2012. Calcium-based functionalization of carbon nanostructures for peptide immobilization in aqueous media. Journal of Materials Chemistry, 22(37), 19684-19693.
Cazorla, C. 2010. Ab initio study of the binding of collagen amino acids to graphene and A-doped (A= H, Ca) graphene. Thin Solid Films, 518(23), 6951-6961.
Gowtham, S., Scheicher, R. H., Ahuja, R., Pandey, R., Karna, S. P. 2007. Physisorption of nucleobases on graphene: Density-functional calculations. Physical Review B, 76(3), 033401.
Varghese, N., Mogera, U., Govindaraj, A., Das, A., Maiti, P. K., Sood, A. K., Rao, C. N. R. 2009. Binding of DNA nucleobases and nucleosides with graphene. ChemPhysChem, 10(1), 206-210.
Ferrari, M. 2005. Cancer nanotechnology: opportunities and challenges. Nature Reviews Cancer, 5(3), 161-171.
Chi, F. L., Guo, Y. N., Liu, J., Liu, Y. L, Huo, Q.S. Size-Tunable and Functional Core−Shell Structured Silica Nanoparticles for Drug Release. 2010. J. Phys. Chem. 114, 2519−2523.
Kuila, T., Bose, S., Khanra, P., Mishra, A. K., Kim, N. H., Lee, J. H. 2011. Recent advances in graphene-based biosensors. Biosensors and Bioelectronics, 26(12), 4637-4648.
Feng, L., Liu, Z. 2011. Graphene in biomedicine: opportunities and challenges. Nanomedicine, 6(2), 317-324.
Chen, F., Liu, S., Shen, J., Wei, L., Liu, A., ChanPark, M. B., Chen, Y. 2011. Ethanol-assisted graphene oxide-based thin film formation at pentane–water interface. Langmuir, 27(15), 9174-9181.
Şenel, M. C., Gürbüz, M., Koç, E. 2015. Gafen Takviyeli Alüminyum Matrisli Yeni Nesil Kompozitler. Engineer & the Machinery Magazine, (669).
Vovusha, H., Sanyal, S., Sanyal, B. 2013. Interaction of nucleobases and aromatic amino acids with graphene oxide and graphene flakes. The Journal of Physical Chemistry Letters, 4(21), 3710-3718.
Rao, C. E. E., Sood, A. E., Subrahmanyam, K. E., Govindaraj, A. 2009. Graphene: the new two-dimensional nanomaterial. Angewandte Chemie International Edition, 48(42), 7752- 7777.
Compton, O. C., Nguyen, S.T. 2010. Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials. small, 6(6), 711-723.
Zhu, Y., Murali, S., Cai, W., Li, X., Suk, J. W., Potts, J. R., Ruoff, R. S. 2010. Graphene and graphene oxide: synthesis, properties, and applications. Advanced materials, 22(35), 3906-3924.
Stankovich, S., Piner, R. D., Nguyen, S. T., Ruoff, R. S. 2006. Synthesis and exfoliation of isocyanatetreated graphene oxide nanoplatelets. Carbon,44(15), 3342-3347.
Oh, J., Lee, J. H., Koo, J. C., Choi, H. R., Lee, Y., Kim, T., ... & Nam, J. D. 201). Graphene oxide porous paper from amine-functionalized poly (glycidyl methacrylate)/graphene oxide core-shell microspheres. Journal of Materials Chemistry, 20(41), 9200-9204.
Kim, S. G., Park, O. K., Lee, J. H., Ku, B. C. 2013. Layer-by-layer assembled graphene oxide films and barrier properties of thermally reduced graphene oxide membranes. Carbon letters, 14(4), 247-250.
Feng, H., Wang, X., Wu, D. 2013. Fabrication of spirocyclic phosphazene epoxy-based nanocomposites with graphene via exfoliation of graphite platelets and thermal curing for enhancement of mechanical and conductive properties. Industrial & Engineering Chemistry Research, 52(30), 10160-10171.
Dang, T. T., Pham, V. H., Hur, S. H., Kim, E. J., Kong, B. S., & Chung, J. S. 2012. Superior dispersion of highly reduced graphene oxide in N, N-dimethylformamide. Journal of colloid and interface science, 376(1), 91-96.
Pei, S., & Cheng, H. M. 2012. The reduction of graphene oxide. Carbon,50(9), 3210-3228.
Worthington-Enzyme Manual. 1972. Enzymes, Enzyme Reagents, related Biochemicals, Xanthineoxidase, Worthington Biochemical Corporation in Freehold, New Jersey, U.S.A. s. 216.
Thema, F. T., Moloto, M. J., Dikio, E. D., Nyangiwe, N. N., Kotsedi, L., Maaza, M., Khenfouch, M. 2012. Synthesis and characterization of graphene thin films by chemical reduction of exfoliated and intercalated graphite oxide.Journal of chemistry, 2013.
Peng, S., Fan, X., Li, S., Zhang, J. 2013. Green synthesis and characterization of graphite oxide by orthogonal experiment. Journal of the Chilean Chemical Society, 58(4), 2213-2217.
Lai, Q., Zhu, S., Luo, X., Zou, M., & Huang, S. 2012. Ultraviolet-visible spectroscopy of graphene oxides. AIP Advances, 2(3), 032146.
Liu, C., Hu, G., Gao, H. 2012. Preparation of fewlayer and single-layer graphene by exfoliation of expandable graphite in supercritical N, Ndimethylformamide. The Journal of Supercritical Fluids, 63, 99-104.
Sahoo, S. K., & Mallik, A. 2015. Simple, fast and cost-effective electrochemical synthesis of few layer graphene nanosheets. Nano, 10(02), 1550019.
Marcano, D. C., Kosynkin, D. V., Berlin, J. M., Sinitskii, A., Sun, Z., Slesarev, A., Tour, J. M. 2010. Improved synthesis of graphene oxide. ACS nano,4(8), 4806-4814.
Roles of the enzyme xanthine oxidase and its products.http://flipper.diff.org/app/pathways/ 3895 (Erişim Tarihi: 22.04.2016).
Harrison, R. 200). Milk xanthine oxidase: Properties and physiological roles. International Dairy Journal, 16(6), 546-554.
Parks, D. A., Granger, D. N. 1986. Xanthine oxidase: biochemistry, distribution and physiology. Acta physiologica Scandinavica. Supplementum, 548, 87.
Kenan, T.W., Patton, S. 1995. The structure of milk: implications for sampling and storage. ss 5-50s. Jensen, R.G., ed. 1995. Handbook of Milk Composition, New York, Academic Pres, U.S.A, 5- 50s.
Dikmen, N., Özgünen, T. ed.1996. Harper' ın Biyokimyası, Barış Kitabevi, İstanbul, 124s.
Massey, V., Brumby, P. E., Komai, H., & Palmer, G. 1969. Studies on milk xanthine oxidase Some spectral and kinetic properties. Journal of Biological Chemistry, 244(7), 1682-1691.
Battelli, M. G., Polito, L., Bolognesi, A. 2014. Xanthine oxidoreductase in atherosclerosis pathogenesis: Not only oxidative stress. Atherosclerosis, 237(2), 562-567.
Zhang, J., Zhang, F., Yang, H., Huang, X., Liu, H., Zhang, J., Guo, S. 2010. Graphene oxide as a matrix for enzyme immobilization. Langmuir, 26(9), 6083-6085.
Liu, Y., Li, Q.,Feng, Y. Y., Ji, G. S., Li, T. C., Tu, J., & Gu, X. D. 2014. Immobilisation of acid pectinase on graphene oxide nanosheets. Chemical Papers, 68(6), 732-738.
Wei, X. L., & Ge, Z. Q. 2013. Effect of graphene oxide on conformation and activity of catalase. Carbon, 60, 401-409.
Grafen. https://tr.wikipedia.org/wiki/Grafen (Erişim Tarihi: 20. 04. 2016).
Chung, C., Kim, Y. K., Shin, D., Ryoo, S. R., Hong, B. H., & Min, D. H. 2013. Biomedical applications of graphene and graphene oxide. Accounts of chemical research, 46(10), 2211-2224.