| Summary: | 博士 === 國立臺灣科技大學 === 化學工程系 === 106 === 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.
|
|---|
