本研究首先探討生物緩衝溶劑(TRIS，TAPS，TES)對溶菌酶穩定性的影響，我們進行了各種生物物理技術分析，如紫外線可見光、螢光、傅里葉變換紅外光譜（FTIR）和圓二色光譜（CD）和動態光散射（DLS）。研究結果顯示這三種緩衝劑即使在較高溫度下也能保持溶菌酶的穩定性，且在較高濃度的緩衝溶液中穩定溶菌酶的結構，在這三種緩衝劑中，TRIS和TES對溶菌酶分子來說是較好的穩定劑，而TAPS緩衝液則是一種相對較弱的穩定劑。
本研究也擇定生物緩衝劑對鳳梨蛋白酶（BM）穩定性的影響為另一探討的主題，通過各種光譜技術瞭解TRIS、TAPS和TES緩衝劑對BM結構的影響。BM酶的穩定性取決於緩衝液的濃度，利用各種生物物理技術，發現BM結構在不同濃度的緩衝劑下，折疊狀態仍能保持完整。結果表明這三種緩衝劑中，TAPS是BM結構的最佳穩定劑，TES是中等穩定劑，TRIS則是弱穩定劑。
我們也透過各種生物物理技術進行血紅蛋白（Hb）與緩衝劑在不同濃度下分子間相互的作用探討。螢光光譜分析的結果表明添加這些生物緩衝劑可增加Hb色氨酸周圍的疏水性，Hb的熱穩定轉變溫度（Tm）隨著生物緩衝液濃度的增加而逐漸增加，實驗證實生物緩衝劑對Hb的結構有影響。三種緩衝液中，TRIS是Hb最強的穩定劑，TES是中等穩定劑，TAPS是弱穩定劑。
此外，我們也探討了在普通緩衝磷酸鹽緩衝液（PBS）存在下，溶菌酶、鳳梨蛋白酶(BM)、血紅蛋白(Hb)和人血清白蛋白（HSA）的穩定性，將每種蛋白質以不同濃度加入到PBS緩衝溶液中，所有樣品均採用各種生物物理技術進行分析。分析結果表明在施加外部因子下，PBS緩衝溶液對酶/蛋白質結構具有很強的保護作用。
分析結果證實本研究所使用的緩衝劑對所選擇的蛋白質/酶均有穩定劑的作用，這些緩衝液對蛋白質/酶均具有保護的能力。然而，在加入更高濃度的PBS緩衝溶液的情況下，Hb結構會受到干擾，這證實緩衝液的類型和濃度會對蛋白質/酶的穩定性有不同程度的影響。本研究表明所擇定的緩衝溶液是具生物相容性的溶劑，可以有效提高蛋白質的穩定性，適於替代傳統揮發性有機化合物和昂貴的新型溶劑。

Abstract

In the present study, the effects of biological buffers including tris (hydroxymethyl) aminomethane (TRIS), N-[tris (hydroxymethyl) methyl]-3-aminopropanesulfonic acid (TAPS), and N-[tris (hydroxymethyl) methyl]-2-aminoethanesulfonic-acid (TES) on the stability of lysozyme have been investigated. To explore the interferences between lysozyme and buffer system, we have performed various biophysical techniques such as UV-visible, fluorescence, Fourier transforms infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Our results reveals that these three buffers enhance the lysozyme thermal stability even at higher temperatures and stabilize the lysozyme structure at higher concentrations of the buffer solutions. Among these three buffers, TRIS and TES buffers are better stabilizers for the lysozyme molecule and the TAPS buffer is a relatively poor stabilizer.
The effects of the selected biological buffers on the stability of bromelain (BM) enzyme are also studied in this work. This study delineates the influences of TRIS, TAPS and TES buffers on the structure of BM by various spectroscopic techniques. The stability of the BM enzyme is dependent on the concentrations of buffers. Using various biophysical techniques, we found that that the folded state of BM structure remains intact in the different concentrations of the studied buffers. The results illustrate that among these three buffers, TAPS buffer is the best stabilizer for BM structure, TES buffer is a moderate stabilizer and the TRIS buffer is a weak stabilizer.
Furthermore, the biomolecular interactions between hemoglobin (Hb) and the selected biological buffers at different concentrations have been investigated by using various biophysical techniques. Fluorescence results reveal that the addition of biological buffers increases the hydrophobicity around the tryptophan environment of Hb. The thermal stability of Hb i.e., the transition temperature (Tm), were found to be gradually increases with increasing the concentrations of the biological buffers. The experimental evidences also reveal that biological buffers have influences on the Hb’s structure. Out of these three buffers, TRIS is the strongest stabilizer for Hb, while TES buffer is a moderate and TAPS buffer is a weak stabilizer.
Additionally, we also explored the stability of lysozyme, BM, Hb and human serum albumin (HSA) in presence of common buffer phosphate buffer saline (PBS). Each protein were added into the PBS buffer solutions at different concentrations. All the samples were analyzed with various biophysical techniques. The analysis results show that PBS buffer solution has great ability to protect enzyme/proteins structures from external additive factors.
The results of the analyses proved that all the selected buffers behave as stabilizers for the selected protein/enzymes. This confirms the potentiality of all these buffers are protecting agents for the proteins/enzymes. However, in case of Hb structure perturbed with the addition of higher concentrations of PBS buffer solution. It also established that the type and concentrations of buffer can effectively influences on the protein/enzyme stability. Generally, our study reveals that the selected buffer solutions are more suitable as biocompatible solvents for the structure and can enhances the stability of proteins. The present study is very useful for providing the alternative media to replace traditional volatile organic compounds or expensive modern solvents for biochemical applications.

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