Polikatvon-protein etkileşiminin ve makromoleküller arası kompleks oluşumunun incelenmesi
Başlık çevirisi mevcut değil.
- Tez No: 66408
- Danışmanlar: PROF. DR. ABDÜLKADİR SEZAİ SARAÇ
- Tez Türü: Doktora
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Polikatyon, Proteinler, Polycation, Proteins
- Yıl: 1997
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Kimya Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
ÖZET Bu çalışmada poli (4-vinilpiridin) (PVP) ' nin etil- bromür ve setilbromür ile kuaternizasyonu sonucunda elde edilen poli (N-etil-vinilpiridinyum bromür) (PEVP) ve poli (N-etil/setil-4-vinilpiridinyuırt bromür) (PECVP) ile Boğa Serum Albumin (Bovine Serum Albumin) (BSA) ve İnsan Serum Albumin (Human Serum Albumin) (HSA) arasındaki etkileşmeler ve makromoleküller arası kompleks oluşumu incelenmiştir. Protein konsantrasyonu sabit tutularak (5.7 uM) artan oranda polimer ilave edilmesi ile hazırlanan karışımların, türbidite ölçümleriyle faz durumları karşılaştırmalı olarak belirlenmiştir. Protein-PECVP sisteminin aksine, başlangıç koşullarında çözünmez haldeki pro-ein-PEVP sisteminin, düşük moleküler tuz konsantrasyonu ve zamana karşı çözeltilerin karıştırılmasıyla çözünür hale geçtiği bulunmuştur. Karıştırma etkisiyle çözünür hale geçen PEVP-protein çözeltilerinin floresans analizinde; çözünür sistemde oluşan polikompleksin karakterinin mol oranıyla değişimi incelenmiştir. Hem tuz hem de karıştırma etkisiyle hetorojen faz durumu değişmeyen PECVP-protein sistemleri santrifüj edilerek elde edilen çözeltiler absorbsiyon ve floresans ölçümleriyle, çökeltiler ise FT-IR spektrumları alınarak incelenmiştir. Kondüktometrik ve potansiyometrik titrasyonlarla, çözünen protein-polimer sistemlerinde elektrostatik etkileşimlere ve makromoleküler kompleks oluşumuna ait titrasyon eğrileri elde edilmiştir. PECVP nin BSA ve HSA ile hidrofobik etkileşmesi sonucunda proteinlerin konformasyon yapısını önemli ölçüde değiştirdiği floresans ölçümleriyle bulunmuştur. Düşük moleküler tuz etkisinin proteinlerin konformasyon değişikliğine tersinir etkisi tuz titrasyonlarıyla incelenmiştir. Karıştırma etkisiyle çözünür hale geçen PEVP-protein sistemlerinde protein emisyonunun maksimum sönüme uğradığı mol oranında; bir polimer zincirine maksimum sayıda protein bağlandığı polikomplekslerin oluştuğu ve bu polikomplekslerin, NaCl konsantrasyonunun etkisiyle bileşenlerine tamamen parçalanmadığı bulunmuştur.
Özet (Çeviri)
SUMMARY STUDY OF POLYCATIONS-PROTEIN INTERACTIONS AND FORMATION OF INTERMACROMOLECULAR COMPLEXES Protein-polyelectrolyte complexes (PPCs) can play an important role in a variety of chemical and biological processes, such as; protein separation, enzyme stabilization and polymer drug delivery. Also, by studying PPC formation, we may improve our understanding of interactions between proteins and nucleic acid in the transcription process. PPCs can be formed as a result of the interaction of polycation chains with the oppositely charged groups of the protein molecules.lt was found that interaction of serum protein with poly (N-ethyl-4-vinylpyridinium bromide) (PEVP) in aqueous solution results in self assembling of the asymmetrical aggregetes. These interpolyelectrolyte complexes have been used as a model to study nucleotide-protein complexation. It has been discovered that protein antigens in these PPCs reveal high immunogenecity, and immunization of animals with PPCs confer high levels of immunological protection. Moreover the enzymes in such complexes remain active and acquire high chemical stability. PEVP interacts with serum proteins through electrostatic forces, whereas poly (N-ethyl/cetyl-4- vinylpyridinium bromide) (PECVP) loaded with lateral hydrophobic groups has been shown to interact with protein through electrostatic and hydrophobic forces. Compaction of the chains of linear polyelectrolyte and changing the behaviour of PPC induced by hydrophobic cetyl groups have been observed. On the basis of analysis of helical structure of BSA in polycomplex by optical rotatory dispersion, enzyme stability and protein-specificity of antibody formation after injection of this polycomplexes into organism, it has been suggested that complex formation in PEVP-BSA system was not accompanied by protein denaturation. However, conformational changes in proteins were observed for the precipitated complexes with poly (N,N-dimethyl- tetramethylene ammonium bromide). Such conformational changes were also observed for interaction of the proteins XIwith poly (N-vinyl-2-pyrolidone) which is nonionic, but not with the homopolymer, Poly (ethylene oxide). This differences were attributed to the differences in the extent of hydrophobicity of the nonionic polymers. The general structure information has been obtained mainly for the PEVP-BSA complexes using the hydrodynamic (viscosity and fast sedimentation) and light scattering measurements which require high concentration of polyelectrolyte. However, in this case, the picture of binding can be greatly complicated by the association of the polycomplex particles occurring at high concentration of components used in these methods. Therefore, it is necessary to use a method allowing one to work lower polymer concentrations. Such a method is the measurement of the fluorescence intensity of BSA and HSA making it possible to lower the concentration of the polyligand by several orders. The applicability of this method to polymer-protein system is due to fact that BSA and HSA have fluorescence properties. BSA contains two tryptophan residues; one of the Trp of BSA is located deep inside of the globular structure, whereas the other is superficially located and fairly accesible to solvent. On the other hand, HSA contains one tryptophan which is surrounded by layers of an amorphous and permeable protein matrix. Fluorescence tecnique can be used for studying the mechanism of polycations interaction with proteins since the changes in emission intensity of Trp in BSA and HSA show the structural changes associated with polycation binding. In this study, interactions of poly (N-ethyl-4-vinyl pyridinium bromide) (PEVP) and poly (N-ethyl/cetyl-4-vinyl pyridinium bromide) (PECPV) with BSA and HSA were investigated. The influence of NaCl concentration, pH and hydrophobic fragments of polymer on the complex formation were analyzed. The structural model of polycomplexes are discussed. Poly (N-ethyl-4-vinylpyridinium bromide) (PEVP) and poly (N-ethyl/cetly-4-vinylpyridinium bromide) (PECVP) were synthesized and fractionated. Quaternization of poly (4- vinylpyr idine) (PVP) was carried out in methanol at 60°C in sealed bulbs under nitrogen atmosphere. The average degree of quaternization (ft) was determined from the content of bromine and nitrogen in the samples and also using FT-IR spectroscopy by the ratio of absorption bands for uncharged (1600 cm“1) and charged (1640 cm-1) pyridine units. (ft«95 for quaternization by ethyl bromide in PEVP ; ft«10 for. quaternization by cetyl bromide and ft«80 for that by ethyl bromide in PECVP). Xll1. Turbidimetric Titration Turbidimetric titrations of BSA and HSA with PEVP and PECVP at various molar ratios, Ri (Ri=np0LYMER/npRoTEtN), were carried out at pH 7.0 and pH 4.3. The increase in the polymer content of the mixture (concentration of BSA and HSA in mixture are constant, nBsA and nHsa=5. 7 uM) leads to the increase in turbidity at pH 7.0. This is due to the initial formation of insoluble polycomplexes. The curves of PEVP-BSA and PEVP-HSA systems display a turbidity maxima at Ri=0.07, whereas that of PECVP-BSA and PECVP-HSA systems at Ri=0.14 and Ri=0.2, respectively. Turbidity at 400 nm (A4oo) is not observed for all mixtures of polymer- protein solutions at pH 4.3. The further increase in the polymer content of the mixture decreases the optical density because of the formation of soluble polymer-protein complexes and simultaneously the insoluble complexes at pH 7.0. The systems become homogeneous when the ratios of the components (Ri) in solution are reached to 0.35 for PEVP- BSA system and 0.56 for PEVP-HSA system. Phase state of the mixture remains unchanged at the further increase of polymer content, whereas the homogeneity of PECVP-BSA and PECVP-HSA mixtures occur at Ri=0.56. The turbidimetric behaviour for the mixtures of polymer-protein systems were analogous and at a proper ratio, the insoluble complexes transform to soluble state. La The Effect of Salt and Stirring The essential difference behaviours of these systems were also observed in the presence of low molecular salt (NaCl) and by stirring on time. Optical density, at 400 nm, of the PEVP-BSA and PEVP-HSA systems decrease with time and insoluble mixtures become homogeneous. On the contrary, the values of A40o is changed insignificantly and the phase state of the reaction mixture remains unchanged for both PECVP-BSA and PECVP-HSA systems. The mixtures of polymer-protein at Ri=0.14 were titrated with solid NaCl to obtain the solutions at different salt concentrations. Starting with 0.05 M NaCl concentration, insoluble PEVP-BSA and PEVP-HSA mixtures became homogeneous and transformed to soluble state followed by the turbidity decrease. The turbidity of both PECVP-BSA and PECVP-HSA systems decreased also, but the transformation of insoluble mixtures to soluble state never took place with increasing salt concentration. xmThe absorbtion spectrum of PEVP-BSA and PEVP-HSA systems were obtained for Ri=0.07 after the solution transformed to homogeneous state. The value of absorbance at 254 ran, which is characteristic for the absorbance of pyridine ring, is lower than that of polymer alone because of the binding of the polymer to protein. The mixtures of PECVP-BSA and PECVP-HSA solutions, which are insoluble at pH 7.0, were centrifuged and then investigated by spectrophotometric methods. Optical density at 280 nm and emission intensity at 343 nm measurements showed that the mixtures of PECVP-BSA solutions at 0.035 <Ri< 007 contain only free protein but the mixtures of PECVP-BSA at 0.07<Ri<0.21 do not contain both polymer and protein. On the other hand, supernatants of PECVP-HSA mixtures contain free protein at 0.035<Ri<0.14. 2. Fluorescence Analysis The mixtures of PEVP-BSA and PEVP-HSA at different Ri were stirred to obtained homogeneous solutions and then the fluorescence spectra, excited at a wavelength of 290 nm, both excitation and emission slits being 5 nm, were recorded. In this experiment the BSA and HSA concentrations were in all cases 5.7 uM. As the concentration of polymer in the mixture is increased, the emission intensity of solution decreased, passed through a minimum at Ri=0. 1 and increased again beyond Ri=0.2, then reached to constant value. The decrease in emission intensity is not due to energy transfer but rather to a conformational effect that changes the excited singlet-state decay rate. The reason for quenching of emission intensity is the binding of protein molecules to the polymer chain. This shows that the maximum numbers of proteins bound to polymer at about Ri=0. 1 because of the maximum reduction of fluorescence intensity. The fluorescence intensity of BSA and HSA are less quenched upon addition of PEVP at pH 4.3. On the contrary, the mixtures of PECVP-BSA and PECVP-HSA at the same mole ratios and the same pH exhibit more quenching results. Therein, the quenching effect of PECVP on emission intensity of BSA and HSA in acidic solution is due to the interaction of protein molecules with polycation chain which is loaded with hyrophobic fragments, at the same time, due to a progressive protein conformational change induced by complexation. Spector and Fletcher have found that the strength of binding. increases as the length of the alkyl chain of a fatty acid increases, suggesting that XIVthe main energy of binding is due to hydrophobic interactions. 2.a The Effect of the Br”on Quenching The precence of bromide ions in polymer might also be important in quenching of protein, but this possibility is considered negligible when we take into account the effect of bromide ions in quenching of fluorescence. Because the concentration of bromide ions in the maximum amount of polymer are not more than 7.6xl0~3 M. 2.b The Effect of The Low Molecular Salt The salt titration of BSA, HSA and the mixture of PEVP-BSA and PEVP-HSA, after homogeneous solution was obtained at pH 7.0, were performed by adding of solid NaCl and stirring until they were dissolved and then the emission intensities at 343 nm were measured. Our fluorescence measurements showed that the largest increases in fluorescence intensities of PEVP-BSA and PEVP-HSA mixtures were observed at about 0.1 M NaCl concentration. The further increasing of salt concentration did not change the intensity of the fluorescence. It can be suggested that NaCl weakens the electrostatic interactions between oppositely charged polycations and protein molecules. This effect can be explained by the influence of low molecular salt on the interaction of polymers with protein globules. On the other hand, the emission intensity of polymer-protein solution at the maximum NaCl concentration is lower than that of free protein since polymer-protein complex at these conditions is not completely decomposed into its components. (NaCl concentration was not effect the emission intensity of BSA and HSA in phosphate buffer, pH 7.0). In this study, we have demonstrated that fluorescence spectroscopy is useful in elucidating the structural and binding properties of serum albumins complexed with polycations. xv
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Başlık çevirisi yok
DİLEK LEMİOĞLU
Yüksek Lisans
Türkçe
1994
Gıda MühendisliğiAnkara ÜniversitesiGıda Mühendisliği Ana Bilim Dalı
PROF. DR. MUAMMER KAYAHAN
