Cibacron Blue F3GA ile modifiye polimerik mikroküreler ile RuBisCO adsorpsiyonu

Öz

Bu çalışmada, Cibacron Blue F3GA (CB) ile modifiye edilmiş poli(hidrokisetil metakrilat) [PHEMA] mikroküreler hazırlandı ve ribuloz-1,5-bisfosfat karboksilaz/oksijenaz (RuBisCO) adsorpsiyonunda kullanıldı. PHEMA mikroküreler süspansiyon polimerizasyonu tekniği ile sentezlendi ve taramalı elektron mikroskopisi (SEM) ve Fourier transform infrared spektroskopisi (FTIR) ile karakterize edildi. CB ile kovalent olarak modifiye edilen CB-PHEMA mikroküreler RuBisCO adsorpsiyonunda kullanıldı. Adsorpsiyona CB içeriği, başlangıç RuBisCO derişimi (0,2-1.0 mg/mL), pH (5,5-9.0), sıcaklık (4°C, 22 oC ve 35°C) ve temas süresinin etkisi araştırıldı. CB-PHEMA mikrokürelerin CB içeriği elementel analiz ile belirlenen %N miktarı kullanılarak hesaplandı. CB-PHEMA mikrokürelere adsorplanan maksimum RuBisCO miktarı 33,59 mg/g olarak belirlendi (22 oC, pH 6,0). Üç farklı sıcaklıkta (4°C, 22 oC ve 35°C) elde edilen RuBisCO adsorpsiyon verilerinin izoterm ve kinetik modellere uygunluğu araştırıldı. RuBisCO adsorpsiyon verilerinin Freundlich izoterm modeli ve yalancı-ikinci derece kinetik modele uygun olduğu belirlendi. RuBisCO adsorpsiyonuna ilişkin Gibbs serbest enerji değişimi (ΔG ͦ) değerleri; 4 oC, 22 oC ve 35 oC sıcaklık için sırasıyla -9,22 kj/mol, -10,44 kJ/mol ve -11,33 kJ/mol olarak hesaplandı. Entalpi değişimi (ΔH ͦ) +9,661 kJ/mol ve entropi değişimi (ΔS ͦ) +68,14 J/mol.K olarak belirlendi. Termodinamik parametreler, CB-PHEMA mikrokürelere RuBisCO adsorpsiyonunun endotermik ve kendiliğinden gerçekleşen bir proses olduğunu kanıtladı. CB-PHEMA mikrokürelerin ıspanak protein ekstraktından RuBisCO adsorpsiyon performansı SDS-PAGE analizi ile değerlendirildi.

Anahtar Kelimeler

• RuBisCO
• adsorpsiyon
• Cibacron Blue F3GA
• bitki proteom analizi

RuBisCO adsorption via polymeric microbeads modified with Cibacron Blue F3GA

Öz

In this study, poly(hydroxyethyl methacrylate) [PHEMA] microbeads modified with Cibacron Blue F3GA (CB) were prepared and used for the adsorption of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). PHEMA microbeads were synthesized by suspension polymerization and characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Then, CB modified CB-PHEMA microbeads were used for RuBisCO adsorption. Effects of CB content, initial RuBisCO concentration (0.2-1.0 mg/mL), pH (5.5-9.0), temperature (4 oC, 22 oC and 35°C) and contact time were investigated. The CB amount of the CB-PHEMA microbeads was calculated via N% determined by elemental analysis. The maximum RuBisCO amount adsorbed onto the CB-PHEMA microbeads was determined as 33.59 mg/g (22 oC, pH 6.0). The RuBisCO adsorption data obtained at three different temperatures (4°C, 22 oC and 35°C) were applied to isotherm and kinetic models. Freudlich isotherm model and pseudo-second order kinetic model were well fitted to adsorption data with high correlation coefficients. Gibss free energy changes (ΔG ͦ) of RuBisCO adsorption for 4 oC, 22 oC and 35 oC were calculated as -9.22 kj/mol, -10.44 kJ/mol and -11.33 kJ/mol, respectively. Enthalpy change (ΔH ͦ) was +9.661 kJ/mol and entrophy change (ΔS ͦ) was +68.14 J/mol.K. The calculated thermodynamic parameters showed that RuBisCO adsorption onto CB-PHEMA microbeads was endothermic and spontaneous. The RuBisCO adsorption performance of CB-PHEMA microbeads from spinach protein extract was evaluated by SDS-PAGE analysis.

Anahtar Kelimeler

• RuBisCO
• adsorption
• Cibacron Blue F3GA
• plant proteomics

Kaynakça

1. Cánovas, F.M., Dumas-Gaudot, E., Recorbet, G., Jorrin, J., Mock, H.P., Rossingnol, M., Plant proteome analysis, Proteomics, 4, 285-298, (2004).
2. Rabilloud, T., Solubilization of proteins for electrophoretic analyses, Electrophoresis, 17,813–829. 1996.
3. Rose, J.K., Bashir, S., Giovannoni, J.J., Jahn, M.M., Saravanan, R.S., Tackling the plant proteome: practical approaches, hurdles and experimental tools Plant Journal, 39,715–733, (2004).
4. des Francs, C.C., Thiellement, H., de Vienne, D., Analysis of leaf proteins by two-dimensional gel electrophoresis: protease action as exemplified by Ribulose bisphosphate carboxylase/oxygenase degradation and procedure to avoid proteolysis during extraction, Plant Physiology, 78,178–182, (1985).
5. Gegenheimer, P., Preparation of extracts from plants, Methods in Enzymology,182,174–193, (1990).
6. Wilkins, M.R., Gasteiger, E., Sanchez, J.C., Bairoch, A., Hochstrasser, D.F. Two-dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number, Electrophoresis, 19,1501– 1505, (1998).
7. Corthals, G.L., Wasinger, V.C., Hochstrasser, D.F., Sanchez, J.C., The dynamic range of protein expression: a challenge for proteomic research, Electrophoresis, 21,1104–1115, (2000).
8. Freeby, S, Berkelman, T, Paulus, A, Liu, N, Wehr, T, Academia, K, Walker, J. 2D-analysis of leaf protein samples treated with proteominer beads under denaturing and non-denaturing conditions. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6441.pdf (12.01.2021)

9. Kim, S.T., Cho, K.S., Jang, Y.S., Kang, K.Y., Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays, Electrophoresis, 22, 2103–2109, (2001).
10. Xi, J.H., Wang, X., Li, S.Y., Zhou, X., Yue, L., Fan, J., Hao, D.Y., Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome, Phytochemistry, 67, 2341–2348, (2006).
11. Cellar, N.A., Kuppannan, K., Langhorst, M.L., Ni, W., Xu, P., Young, S.A., Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphatecarboxylase/oxygenase, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 861, 29–39, (2008).
12. Denizli, A., and Piskin, E., Dye-ligand affinity systems, Journal of Biochemical and Biophysical Methods, 49, 391–416, (2001).
13. Stellwagen, E., Chromatography on immobilised reactive dyes, Methods in Enzymology,182, 343–357, (1990).
14. Roschlau, P., Hess, B., Affinity chromatography of yeast pyruvatekinase with Cibacron blue bound to Sephadex G-200, Hoppe-Seyler's Zeitschrift fur physiologische Chemie, 353,441–443, (1972).
15. Lowe, C.R., Gald, M., Larsson, P.O., Ohlson, S., Small, D.A.P., Atkinson, T., High performance liquid affinity chromatography of proteins on Cibacron Blue F3GA bonded silica, Journal of Chromatography, 215-303, (1981).
16. Lowe, C.R., Hans, M., Spibey, N., Drabble, W.T., The purification of inosine-5-monohosphate dehydrogenase from Escherichia coli by affinity chromatography on immobilized Procion dyes, Analytical Biochemistry, 104, 23–28, (1980).
17. Dean, P.D.G., Watson, D.H., Protein purification using immobilised triazine dyes, Journal of Chromatography,165: 310–319, (1969).
18. Bollin, E., Vastola, K., Olezsak, P., Sulkowski, E., The interaction of mamallian interferons with immobilized Cibacron Blue F3GA: modulation of binding strength, Preparative Biochemistry, 8, 259–264, (1978).
19. Biellman, J.F., Samma, J.P., Branden, C.I., Eklund H., X-ray studies of the binding of Cibacron Blue F3GA to liver alcohol dehydrogenase, European Journal of Biochemistry,102-107, (1979).
20. Travis, J., Pannell, R., Selective removal of albumin from plasma by affinity chromatography, Clinica Chimica Acta, 49, 49-52, (1973).
21. Travis, J., Brown, J., Tewksbury, D., Johnson, D., Pannell, R., Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma, Biochemical Journal,157, 301-306, (1976).
22. Odabaşı, M., Denizli, A., Cibacron Blue F3GA incorporated magnetic poly(2-hydroxyethyl methacrylate) beads for lysozyme adsorption, Journal of Applied Polymer Science, 93, 719–725, (2004).
23. Jankowski, W.J., Ansen, W.H., Sulkowski, E., Carter, W.A., Binding of human interferons to immobolized Cibacron Blue F3GA: The nature of molecular interaction. Biochemistry, 15, 5182-5187, (1976).
24. Nynäs, A.L., White proteins from green leaves in food applications. A literature study. Alnarp: Sveriges lantbruksuniversitet. (Introductory paper at the Faculty of Landscape Architecture, Horticulture and Crop Production Science, (2018).
25. Langmuir, I., The constitution and fundamental properties of solids and liquids. Part 1. Solids, Journal of the American Chemical Society, 38, 2221 – 2295, (1916).
26. Freundlich, H.M.F., Over the Adsorption in Solution, Journal of Physical Chemistry, 57, 385–471, (1906).
27. Dubinin, M.M., Radushkevich, L.V., The equation of the characteristic curve of activated charcoal, Proceedings of the Academy of Sciences of the USSR. Physical Chemistry Section, 55,331–337, (1947).
28. Unlü, N., Ersoz M., Adsorption characteristics of heavy metal ions onto a low cost biopolymer sorbent from aqueous solutions, Journal of Hazardous Materials, 136, 272–280, (2006).
29. Lagergren, S., Zur theorie der sogenannten Adsorption gel oster stoffe, Kungliga Svenska Vetenskapsakademiens, Handlingar, 25,4, 1–39, (1898).
30. Ho, Y.S., McKay, G., Pseudo-second-order model for sorption processes, Process Biochemistry, 34, 451–465, (1999).
31. Ellis, R.J., Most abundant protein in the World, Trends in Biochemical Sciences, 4, 241-244, (1979).
32. Vapaavuori, E.M., Correlation of activity and amount of ribulose 1,5-bisphosphate carboxylase with chloroplast stroma crystals in water-stressed willow leaves, Journal of Experimental Botany, 37, 174, 189-198, (1986).
33. Maeda, N., Kitano, K., Fukui, T., Ezaki, S., Atomi, H., Miki, K., Tadayuki Imanaka, T., Ribulose bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon pyrococcus kodakaraensis kod1 is composed solely of large subunits and forms a pentagonal structure, Journal of Molecular Biology, 293, 57-66, (1999).
34. Yang, H.,Wang, G., Zhang, T., Beattie, J.H., Zhou S. Establishing an optimized method for the separation of low and high abundance blood plasma proteins. Analytical Chemistry at Peer Journal. DOI: 10.7717/peerj-achem.6, (2020).
35. Gupta R. High abundance proteins: Proteomer’s thorns in the flesh? Journal of Proteomics & Bioinformatics. 10, 7, 1000e35,(2017).
36. Zolotarjova, N., Nicol, G., Martosella, J., Yang, L-S., Zhang, K., Boyes, B., Evaluation of serum albumin depletion methods for proteomic analysis, HPLC, Nice, France, (2003).