Investigating conjugation reactions between different alzheimer's amyloid beta peptide sequences with polyelectrolytes

Term of amyloid has become an important subject of study in the scientific world especially in recent years. The most significant reason for this is the fact that one of the most important factors causing Alzheimer disease is formation of amyloid beta accumulations. Alzheimer disease is resulted from misfolded amyloid beta peptides forming accumulation in the brain in the form of plaques. Such plaques are made up of small peptide sequences consisting of 39-43 amino acids that are referred to as amyloid beta (Aβ). Aβ is formed through cleavage of a bigger protein, which is known as amyloid precursor protein, with the help of various enzymes. The resulted Aβ peptide fragments are deposited outside neurons to create structures, which are called as senile plaques. The object of this thesis study is to develop peptide-macromolecule bioconjugates and physical complexes having biological activity that can be used in the fields like development of a peptide-based vaccine or diagnostic kit etc for Alzheimer disease, which is considered as one of the most important diseases of our era. 2 different amino acid sequences of amyloid beta peptide (Aβ) with antigenic feature were purchased commercially. Both of the peptide sequences were joined in 4 different carrier macromolecules covalently separately through different mechanisms and at different degrees and furthermore, their physical complexes with these carriers were obtained. Macromolecules used in this study are: Polyacrylic acid (PAA), [Poly(N-vinyl-2-pyrrolidone-co-acrylicacid) or VPAA], Dextran aldehyde (DA) and Bovine Serum Albumin (BSA). PAA and BSA of these carriers were purchased commercially while VPAA and DA were synthesized and characterized in our department. Both of the peptide sequences were equipped with tryptophan amino acid at the amino terminal to make them fluorescent to allow examining the peptides and the bioconjugates to be synthesized under a fluorescent spectrophotometer. Physicochemical nature of all synthesized bioconjugates and complexes were investigated and they were characterized. Formation of bioconjugates and complexes was enlightened through different spectroscopic and chromatographic methods. Comparing the produced results, it was observed that the bioconjugates, which had been produced through covalent bonds in aqueous solution in presence of carboimide, had been attached through more efficient and stronger links. Fluorescent stain was used during occurrence of conjugation reaction for calculating intermolecular distance with the help of Fluorescent Resonance Energy Transfer (FRET) method and the data produced from calculation of distance between the donor and the acceptor was used to calculation of conformation of the pure peptide and variations in conformation of the peptide during formation of the bioconjugate and they were quantitatively expressed. Furthermore, an amyloid beta (1-21) peptide sequence containing Trp was modeled for the first time in the literature, and its optimized geometry in which it had the minimum energy was identified through the molecular mechanical method. It was justified that numeric values, which were produced based on experimental data through FRET method, were consistent with those, which were calculated theoretically. For the amyloid beta (Aβ) peptide's carrier macromolecule bioconjugates that we designed as a model of synthetic vaccine, it was evidenced that the produced bioconjugates did not demonstrate a significant toxic effect especially at lower doses according to the studies, which were conducted on MCF-7 cells at lower concentrations used in in-vitro applications. Furthermore, IC50 values were calculated by checking cell vitality ratios through the MTT tests conducted with these bioconjugates. On the other hand, in another study included in the thesis conducted with the cell culture, the physical complexes and bioconjugates, which had been synthesized from both of the peptide sequences at different concentrations, the carrier molecules alone and the peptide sequences alone were administered to Human Umbilical Vein Endothelial Cells (HUVECs) through the XCELLienge System, which is a new method taking real time measurements for 72 hours with no need for marking, to investigate these matters' effect on cell proliferation rate and their toxic effects. Comparing effect of the bioconjugate and that of the physical complex on HUVECs, it was observed that there was no significant adverse effect of especially the bioconjugates, which were joined through covalent bonds, on cell proliferation and furthermore, usability of the bioconjugates on lab animals was displayed through the data obtained by cell studies conducted on both of MCF-7 and HUVEC. Thus, this is a study enlightening producing antibodies specific to Alzheimer disease, designing nano-biosensors and development of diagnostic kits and biotechnological vaccines including Aβ conjugates, which were synthesized under the scope of this thesis study. Key words: Amyloid beta peptide, Alzheimer, biotechnological vaccines, bioconjugate, covalent conjugation, adjuvant, carbodiimide, toxicity