| Summary: | 博士 === 中原大學 === 化學研究所 === 102 === The natural clay Montmorillonite is inorganically layered material and possesses lattice defects with negative charges. Neutralization of the positively charged protein molecules by the negative charges of the layered lattices in the clay can be applied to achieve the adsorption and desorption of these protein molecules. Protein solution can be prepared at an optimal pH value comparing its respective isoelectric point (pI). As soon as the environmental pH is less than the pI value, the protein is to be positively charged, and will be adsorbed within the lattice formed template; as soon as the environmental pH is adjusted higher than pI, the protein becomes negatively charged and will be easily desorbed.
Montmorillonite as natural clay was organically modified into “Cocamidopropyl Hydroxysultaine (CS)” and “Cocamidopropyl Betaine (HP)” by two different ion exchange methods, respectively. Change of the space of the lattice layers in the inorganic natural and modified clays can be observed by X-ray diffraction (XRD). Organic and inorganic functional groups of the long carbon chains modified into the montmorillonite layers as CS and HP can be well identified by Fourier Transform Infrared (FT-IR). The quantitative analysis of the intercalation capacity of CS and HP as well as the modified natural clay can be processed by Thermogravimetry analyzer (TGA). The surface charge densities as well as zeta potentials of the natural and modified lattice surfaces can be analyzed. Preparation of CL111-HP was also described here.
Selective adsorptions and desorptions of proteins ─ BSA, Lysozyme, and RFP by means of their characteristic isoelectric points have been investigated using natural (montmorillonite) and modified (CS, HP and CL111-HP) clays. Separation of these proteins has been successfully demonstrated by Fluorescence spectrometry, UV, and SDS PAGE.
BSA adsorption capacities of 259.7 mg/g and 187.3 mg/g, desorption of 95.1 mg/g and 106.0 mg/g in gram of either CS or HP, as well as 46.6 % and 56.6 % on the lattice templates of CS and HP, respectively, have been measured by UV with wavelength 280 nm. The adsorption isotherm Langmuir equation was used for the theoretical comparison. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis of CS and HP mixed with both BSA and lysoyzme solutions was used to demonstrate the desorption ability of BSA comparing that of lysoyzme from both modified clay lattices. Such inorganic layer or lattice materials are well applicable for the protein purification has been demonstrated.
Furthermore, red fluorescent protein (RFP) and CL111-HP also modified from natural clay were used instead of either BSA or lysozyme combined with CS and HP for the fluorescence experiment. Rates of adsorption and desorption were reached and optimized to 94.2 % and 84.7%, respectively. 75 abs./mL was achieved as the best degree of saturation adsorption of RFP. It takes only 10 mins for the optimal adsorption. However, the modified CL111-HP to purify RFP can be used for only once.
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