First purification of glutathione S-transferase enzyme from honey bees (Apis mellifera L.) and effects of heavy metal ions on bioactivity

Abstract

Honey bees (Apis mellifera L.) in the Bingöl province of Türkiye play a crucial role in both pollination and contributing to biological processes through their bioactive compounds. Owing to these functions, they possess anticancer, anti-inflammatory, ecological, and economic significance. Glutathione S-transferases (GSTs; EC 2.5.1.18) are detoxification enzymes found in all cellular organisms, including bacteria, plants, insects, and humans. Purifying the glutathione S-transferase enzyme (GST; EC 2.5.1.18) from Apis mellifera L. and examining how different metal ions affect the activity of the purified enzyme were the objectives of this study. The purification process was performed in two steps: homogenate preparation and glutathione-agarose affinity chromatography. The GST was purified with a specific activity of 0.379 EUmg-1 protein, yielding a 7.7% yield and a 77.2-fold purification. SDS-PAGE analysis was used to verify the purity of the enzyme, and only a single protein band was detected. Metal ion effects were evaluated through activity percentage plots, inhibition constants (Ki), and half-maximal inhibitory concentration (IC₅₀) values. Inhibition types were determined using Lineweaver–Burk plots for the metal ions exhibiting inhibitory effects. Pb²⁺ had no significant effect on enzyme activity. In contrast, Fe²⁺ and Co²⁺ ions activated the enzyme, while Hg²⁺, Ag⁺, and Cu²⁺ showed non-competitive inhibition. To the best of our knowledge, this is the first report on the purification of GST from honey bees.

Keywords

• Bee
• Glutathione S-transferase
• Purification
• Metal ions

Supporting Institution

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Project Number

Bu araştırma kamu, ticari veya kâr amacı gütmeyen sektörlerdeki fon sağlayıcı kuruluşlardan herhangi bir özel hibe almamıştır.

Ethical Statement

This study does not contain any experiments with human participants or animals

Bal arılarından (Apis mellifera L.) glutatyon S-transferaz enziminin ilk saflaştırılması ve ağır metal iyonlarının biyoaktivite üzerine etkileri

Abstract

Bingöl ilindeki bal arıları (Apis mellifera L.) hem çiçeklerin tozlaşmasını sağlar hem de taşıdıkları önemli biyoaktif bileşenlerle biyolojik fonksiyonlara katkıda bulunurlar. Bu önemli rollerinden dolayı antikanser, antiinflamatuar, ekolojik ve ekonomik öneme sahiptirler. Glutatyon transferazlar (EC 2.5.1.18), tüm hücresel organizmalarda (bakteriler, bitkiler, böcekler ve insanlar) bulunan detoksifikasyon enzimleridir. Bu çalışmada ilk kez arı dokularından (Apis mellifera L.) glutatyon S-transferaz enziminin (GST; EC 2.5.1.18) saflaştırılması ve bazı metal iyonlarının enzim aktivitesi üzerine etkilerinin araştırılması hedeflenmiştir. Saflaştırma işlemi, homojenat hazırlama ve glutatyon-agaroz afinite kromatografisi olmak üzere iki adımda gerçekleştirildi. Çalışmada, arı dokularından 0,379 EUmg-1 protein spesifik aktivitesine sahip glutatyon S-transferaz enzimi; % 7,7 verimle 77,2 kat saflaştırıldı. Enzimin saflığı SDS-PAGE ile kontrol edildi ve tek bant elde edildi. Ayrıca bazı metal iyonlarının enzim aktivitesi üzerine etkileri incelendi. Metal iyonları için % activite - [metal iyonu] ve Lineweaver-Burk grafikleri yardımıyla sırası ile IC50 değerleri ve Ki sabitleri hesaplandı ve inhibitör etki gösteren metal iyonları için inhibisyon tipleri belirlendi. Metal iyonlarından Pb+2 iyonu enzim aktivitesi üzerinde herhangi bir etki oluşturmazken, Fe+2, Co+2 iyonları enzimi aktive, Hg+2, Ag+, Cu+2 iyonlarının enzimi yarışmasız olarak inhibe ettikleri tespit edildi.

Keywords

• Arı
• Glutatyon S-transferaz
• Saflaştırma
• Metal iyonlar

Supporting Institution

Bu araştırma kamu, ticari veya kâr amacı gütmeyen sektörlerdeki fon sağlayıcı kuruluşlardan herhangi bir özel hibe almamıştır.

Project Number

Bu araştırma kamu, ticari veya kâr amacı gütmeyen sektörlerdeki fon sağlayıcı kuruluşlardan herhangi bir özel hibe almamıştır.

Ethical Statement

Bu çalışma insan katılımcıları veya hayvanlarla herhangi bir deney içermemektedir

References

1. Guven N, Soydan E. Characterization of glutathione S-transferase enzyme from brown meagre (Sciaena umbra) and inhibitory effects of heavy metals. Biotechnol Appl Biochem. 2022;69(1):145-148.
2. Findlay VJ, Townsend DM, Tew KD. Glutathione and glutathione S-transferases in drug resistance. Cancer Drug Resist. 2006:213-221.
3. Simoni RD, Hill RL, Vaughan M. The discovery of glutathione by F. Gowland Hopkins and the beginning of biochemistry at Cambridge University. J Biol Chem. 2002;277(24):27-28.
4. Aybek H, Temel Y, Ahmed BM, Ağca CA, Çiftci M. Deciphering of the effect of chemotherapeutic agents on human glutathione S-transferase enzyme and MCF-7 cell line. Protein Pept Lett. 2020;27(9):888-894.
5. Han J, Won EJ, Hwang DS, Rhee JS, Kim IC, Lee JS. Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus. Comp Biochem Physiol C Toxicol Pharmacol. 2013;158(2):91-100.
6. Yamamoto K, Zhang P, Miake F, Kashige N, Aso Y, Banno Y, et al. Cloning, expression and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol. 2005;141(3):340-346.
7. Hearne JL, Colman RF. Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1. Protein Sci. 2006;15(6):1277-1289.
8. Temel Y, Taysi MŞ. The effect of mercury chloride and boric acid on rat erythrocyte enzymes. Biol Trace Elem Res. 2019;191(1):177-182.

9. Nikolić TV, Kojić D, Orčić S, Vukašinović EL, Blagojević DP, Purać J. Laboratory bioassays on the response of honey bee (Apis mellifera L.) glutathione S-transferase and acetylcholinesterase to the oral exposure to copper, cadmium, and lead. Environ Sci Pollut Res. 2019;26:6890-6897.
10. Yu X, Sun R, Yan H, Guo X, Xu B. Characterization of a sigma class glutathione S-transferase gene in the larvae of the honeybee (Apis cerana cerana) on exposure to mercury. Comp Biochem Physiol B Biochem Mol Biol. 2012;161(4):356-364.
11. Sharma RK, Agrawal M. Biological effects of heavy metals: an overview. J Environ Biol. 2005;26:301-313.
12. Boyd RS. Heavy metal pollutants and chemical ecology: exploring new frontiers. J Chem Ecol. 2010;36:46-58.
13. van Ooik T, Rantala MJ, Saloniemi I. Diet-mediated effects of heavy metal pollution on growth and immune response in the geometrid moth Epirrita autumnata. Environ Pollut. 2007;145:348-354.
14. van Ooik T, Pausio S, Rantala MJ. Direct effects of heavy metal pollution on the immune function of a geometrid moth Epirrita autumnata. Chemosphere. 2008;71:1840-1844.
15. Dubovskiy LM, Grizanova EV, Ershova NS, Rantala MJ, Glupov VV. The effects of dietary nickel on the detoxification enzymes, innate immunity and resistance to the fungus Beauveria bassiana in the larvae of the greater wax moth Galleria mellonella. Chemosphere. 2011.
16. Conti ME, Botre F. Honeybees and their products as potential bioindicators of heavy metals contamination. Environ Monit Assess. 2001;69:267-282.
17. Celli G, Maccagnani B. Honey bees as bioindicators of environmental pollution. Bull Insectol. 2003;56:137-139.
18. Claudianos C, Ranson H, Johnson RM, Biswas S, Schuler MA, Berenbaum MR, et al. A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee. Insect Mol Biol. 2006;15:615-636.
19. vanEngelsdorp D, Meixner MD. A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. J Invertebr Pathol. 2010;103.
20. Lambert O, Piroux M, Puyo S, Thorin C, Larhantec M, Delbac F, et al. Bees, honey and pollen as sentinels for lead environmental contamination. Environ Pollut. 2012;170:254-259.
21. Johnson RM. Honey bee toxicology. Annu Rev Entomol. 2015;60:415-434.
22. Nikolić TV, Kojić D, Orčić S, Vukašinović EL, Blagojević DP, Purać J. Laboratory bioassays on the response of honey bee (Apis mellifera L.) glutathione S-transferase and acetylcholinesterase to the oral exposure to copper, cadmium, and lead. Environ Sci Pollut Res. 2019;26(7):6890-6897.
23. Armstrong RN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol. 1997;10(1):2-18.
24. Yu X, Sun R, Yan H, Guo X, Xu B. Characterization of a sigma class glutathione S-transferase gene in the larvae of the honeybee (Apis cerana cerana) on exposure to mercury. Comp Biochem Physiol B Biochem Mol Biol. 2012;161(4):356-364.
25. Engwa GA, Ferdinand PU, Nwalo FN, Unachukwu MN. Mechanism and health effects of heavy metal toxicity in humans. In: Karcioglu O, editor. Poisoning in the Modern World: New Tricks for an Old Dog. 2019. p. 77-99.
26. Erat M, Guvercin S, Sakiroglu H. Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes. Protein Pept Lett. 2008;15(1):6-12.
27. Toribio F, Martinez-Lara E, Pascual P, Lopez-Barea J. Methods for purification of glutathione peroxidase and related enzymes. J Chromatogr B Biomed Sci Appl. 1996;684(1-2):77-97.
28. Ahmed BM, Temel Y, Çiftci M. Purification and characterization of glutathione S-transferase enzyme from quail (Coturnix coturnix japonica) heart and investigation of the effect of some metal ions on enzyme activity. Cumhuriyet Sci J. 2019;40(4):802-812.
29. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130-7139.
30. Kakkar P, Jaffery FN. Biological markers for metal toxicity. Environ Toxicol Pharmacol. 2005;19:335-349.
31. 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.
32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. doi:10.1038/227680a0.
33. Enayati A, Ranson H, Hemingway J. Insect glutathione transferases and insecticide resistance. Insect Mol Biol. 2005;14(1):3-8.
34. Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol. 2005;45:51-88.
35. Özaslan MS, Demir Y, Küfrevioğlu OI, Çiftci M. Some metals inhibit the glutathione S-transferase from Van Lake fish gills. J Biochem Mol Toxicol. 2017;31(11).
36. Güller U, Taşer P, Çiftci M, Küfrevioğlu Öİ. Purification of glutathione S-transferase from Bonito (Sarda sarda) liver and investigation of metal ion effects on enzyme activity. Hacettepe J Biol Chem. 2014;42(3):435-442.
37. Caglayan C, Temel Y, Türkeş C, Ayna A, Ece A, Beydemir Ş. The effects of morin and methotrexate on pentose phosphate pathway enzymes and GR/GST/TrxR enzyme activities: an in vivo and in silico study. Arch Pharm (Weinheim). 2024;357(2). doi:10.1002/ardp.202300497.
38. Ayna A, Khosnaw L, Temel Y, Ciftci M. Antibiotics as inhibitor of glutathione S-transferase: biological evaluation and molecular structure studies. Curr Drug Metab. 2021;22(4):308-314. doi:10.2174/1389200222666210118102700.
39. Bayındır S, Ayna A, Temel Y, Çiftçi M. The synthesis of new oxindoles as analogs of natural product 3,3′-bis(indolyl)oxindole and in vitro evaluation of the enzyme activity of G6PD and 6PGD. Turk J Chem. 2018;42(2):332-345. doi:10.3906/kim-1706-51.
40. Temel Y, Ayna A, Shafeeq IH, Ciftci M. In vitro effects of some antibiotics on glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocyte. Drug Chem Toxicol. 2020;43(2):219-223. doi:10.1080/01480545.2018.1481083.
41. Bayındır S, Temel Y, Ayna A, Çiftçi M. The synthesis of N-benzoylindoles as inhibitors of rat erythrocyte glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. J Biochem Mol Toxicol. 2018;32(9). doi:10.1002/jbt.22193.