Polinükleer Metal Komplekslerin Plazmid DNA, Alkalen Proteaz Üzerine Etkileri ve Antioksidan Kapasiteleri

Öz

Bu çalışmada, daha önce sentezlenmiş olan ,N′-Bis[1-(4-fenilfenil)-2-hidroksiimino-2-(pirolidino)-1-etiliden]-1,3-propandiamin ve bunun heterodinükleer Cu(II)-Mn(II) ve Cu(II)-Co(II) kompleksileri, N,N′-Bis[1-(4-fenilfenil)-2-hidroksiimino-2-(4-kloroanilino)-1-etiliden]-1,3-propandiamin ve bunun homodinükleer Cu(II) kompleksi ve homotrinükleer Cu(II) komplekslerinin Bacillus subtilis kaynaklı alkalen proteaz ve plazmid DNA’ya olan etkileri ile antioksidan aktiviteleri araştırılmıştır. Enzim, uygun koşullarda üretildikten sonra amonyum sülfat çöktürme yöntemi ve jel filtrasyon tekniği ile %9,9 verimle ve 69,6 kat saflaştırılmış; SDS-PAGE ile protein molekülünün ağırlığı 30,0 kDa bulunmuştur. Komplekslerin proteaz aktivitesini artırdığı belirlenmiştir. Antioksidan testlerde, homodinükleer Cu(II) içeren B2 kompleksi en yüksek etkiyi göstermiştir. Ayrıca, Cu(II) komplekslerinin DNA’nın doğrusal formunu artırdığı, H₂O₂ varlığında bu etkinlik belirgin şekilde artmıştır. Komplekslerin bazıları tamamen DNA’yı parçalarken bazıları ise hidrojen peroksit içeriğiyle süper sarmal şeklini tümüyle parçalamıştır.

Anahtar Kelimeler

• Alkaline proteaz
• Bacillus subtilis
• Schiff bazı
• Antioksidan
• Plazmid DNA.

Effects of Polynuclear Metal Complexes on Plasmid DNA, Alkaline Protease, and Their Antioxidant Capacities

Öz

In this work, the influence of previously synthesized compounds on alkaline protease isolated from Bacillus subtilis, their interaction with plasmid DNA, and their antioxidant potential were examined.The compounds evaluated included N′-Bis[1-(4-phenylphenyl)-2-hydroxyimino-2-(pyrrolidino)-1-ethylidene]-1,3-propanediamine along with its heterodinuclear Cu(II)-Mn(II) and Cu(II)-Co(II) complexes, as well as N,N′-Bis[1-(4-phenylphenyl)-2-hydroxyimino-2-(4-chloroanilino)-1-ethylidene]-1,3-propanediamine and its homodinuclear and homotrinuclear Cu(II) complexes. Under optimized production conditions, the enzyme underwent purification utilizing a sequential process of ammonium sulfate precipitation followed by gel filtration chromatography. This process led to a 69.6-fold increase in purity, with an overall recovery yield of 9.9%. SDS-PAGE analysis revealed a single protein band with a molecular weight of approximately 30.0 kD. Metal complexes have been demonstrated to enhance protease activity. Among the complexes under investigation, the homodinuclear Cu(II) complex B2 demonstrated the highest antioxidant activity. Furthermore, the presence of Cu(II) complexes resulted in an augmentation of linear plasmid DNA, an effect that was considerably amplified in the presence of H₂O₂. It was observed that certain complexes were capable of completely degrading DNA, while others were able to disrupt the supercoiled structure upon the addition of hydrogen peroxide.

Anahtar Kelimeler

• Alkaline protease
• Bacillus subtilis
• Schiff base
• Antioxidant
• Plasmid DNA.

Kaynakça

1. [1] Adinarayana, K., Ellaiah, P., Prasad, D. S. 2003. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS PharmSciTech, 4(4), 440–448.
2. [2] Patel, R. K., Dodia, M. S., Joshi, R. H., Singh, S. P. 2006. Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochemistry, 41(9), 2002–2009.
3. [3] Zambare, V. 2010. Purification and Characterization of Neutral Serine Protease from Bacillus sp. Asiatic Journal of Biotechnol. Resources, 3, 183–192.
4. [4] Beg, K. Q., Gupta, R. 2003. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology, 32(2), 294–304.
5. [5] Singh, J., Vohra, R. M., Sahoo, D. K. 2001. Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus. Journal of Industrial Microbiology & Biotechnology, 26(6), 387–393.
6. [6] Huang, Q., Peng, Y., Li, X., Wang, H., Zhang, Y. 2003. Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Current Microbiology, 46(3), 169–173.
7. [7] Joo, H. S., Chang, C. S. 2005. Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties. Process Biochemistry, 40(3–4), 1263–1270.
8. [8] Sevinç, N., Demirkan, E. 2011. Production of Protease by Bacillus sp. N-40 Isolated from Soil and Its Enzymatic Properties. Journal of Biology of the Environment Sciences, 5(14), 95–103.

9. [9] Nilegaonkar, S. S., lowry, V. P., Kanekar, P. P., Dhakephalkar, P. K., Sarnaik, S. S., Chopade, B. A. 2007. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresource Technology, 98(7), 1238–1245.
10. [10] Gupta, R., Beg, Q. K., Khan, S., Chauhan, B. 2005. One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp. Journal of Chromatography A, 1075(1–2), 103–108.
11. [11] Johnvesly, B., Naik, G. R. 2001. Production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochemistry, 37(2), 139–144.
12. [12] Dede, B., Karipcin, F., Cengiz, M. 2009. Novel homo- and hetero-nuclear copper(II) complexes of tetradentate Schiff bases: Synthesis, characterization, solvent-extraction and catalase-like activity studies. Journal of Hazardous Materials, 163, 1148–1156.
13. [13] Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.
14. [14] Anson, M.L. (1938) The Estimation of Pepsin, Trypsin, Papain, and Cathepsin with Hemoglobin. The Journal of General Physiology, 22, 79-89.
15. [15] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.
16. [16] Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use Of A Free Radical Method To Evaluate Antioxidant Activity. LWT - Food Science and Technology. Volume 28(1), 25-30.
17. [17] Fukumoto, L.R., & Mazza, G., 2000. Assessing antioxidant and prooxidant activities of phenolic compounds. Journal of agricultural and food chemistry. 48(8), 3597-3604.
18. [18] Mates J M, Perez-Gomez C & Nunez de Castro I (1999). Antioxidant enzymes and human diseases. Clin. Biochem. 32: 595–603.
19. [19] Mei, C., Jiang, X. 2005. A novel surfactant- and oxidation-stable alkaline protease from Vibrio metschnikovii DL 33-51. Process Biochemistry, 40(6), 2167–2172.
20. [20] Yang, J., Shih, I., Tzeng, Y., Wang, S. 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme and Microbial Technology, 26(5–6), 406–413.
21. [21] Reddy, K. H., Reddy, P. S. 2000. Nuclease Activity of Mixed Ligand Complexes of Copper (II) with Heteroaromatic Derivatives and Picoline. Transition Metal Chemistry, 25(5), 505–510.
22. [22] Sağlam, N., Çolak, A., Serbest, K., Dülger, S., Güner, S., Karaböcek, S., Beldüz, A. O. 2002. Oxidative Cleavage of DNA by Homo- and Heteronuclear Cu(II)-Mn(II) Complexes of an Oxime-Type Ligand. Biometals, 15(4), 357–365.
23. [23] Ceyhan, G., Çelik, C., Uruş, S., Demirtaş, İ., Elmastaş, M., Tümer, M. 2011. Antioxidant, electrochemical, thermal, antimicrobial and alkane oxidation properties of tridentate Schiff base ligands and their metal complexes. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 81(1), 184–198.
24. [24] Zhang, Y., Zou, B., Chen, Z., Pan, Y., Wang, H., Liang, H., Yi, X. 2011. Synthesis and antioxidant activities of novel 4-Schiff base-7-benzyloxy-coumarin derivatives. Bioorganic & Medicinal Chemistry Letters, 21(22), 6811–6815.
25. [25] Tian, M., Ihmels, H., Brötz, E. 2010. DNA cleavage by the Cu (ii) complex of the DNA-intercalating 9-bis (pyridin-2-ylmethyl) aminobenzo [b] quinolizinium. Dalton Transactions, 39(35), 8195-8202.
26. [26] Massoud, S. S., Louka, F. R., Xu, W., Perkins, R. S., Vicente, R., Albering, J. H., Mautner, F. A. 2011. DNA cleavage by structurally characterized dinuclear copper (II) complexes based on triazine. European Journal of Inorganic Chemistry, 2011(23), 3469–3479.
27. [27] Kim, J. H., Kim, S. H. 2003. Double Strand DNA Cleavage by a Dinuclear Cu(II) Complex. Chemistry Letters, 32(6), 490.