分子內/分子間雙硫鍵對牛血清白蛋白結構穩定性與類伴護活性之影響

• Main Authors: Chih-Hsien Wang, 王智賢
• Other Authors: Wenlung Chen
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/85439742408314778018

博士 === 國立嘉義大學 === 應用化學系研究所 === 104 === Thiol (sulfhydryl) and disulfide bond play crucial role in maintaining function and structure of protein. BSA contains 17 pairs of intramolecular disulfide bonds and one sulfhydryl. Due to this property, BSA is currently used for studying the effect of disulfide bond on structural stability of protein. Marini et al. reported that BSA has chaperone-like activity which is similar to the function of α-crystallin in 2005. So far, however, scientists have yet understood the reason for BSA to exhibit chaperone-like activity property. Thus, this research demonstrates the conformational and secondary structural transform of BSA during chaperone by utilizing native PAGE and FT-Raman. Results showed that the predominant secondary structure of BSA was the relatively unstable α-helix conformation. Nevertheless, with 17 pairs of intramolecular disulfide bonds to limit the foiling of partial tertiary structure, BSA is able to maintain a comparably stable secondary structure even after the treatment of heating and urea. It is only when the intramolecular disulfide bond is disrupted that it will destroy the folded tertiary structure and consequently exposes the hydrophobic amino acids. As a result, the intramolecular hydrophilic interaction between BSA molecules would be reinforced and eventually lead to aggregation. Heating increases effectively the probability and rate of thiol-disulfide bond exchange reaction hence accelerating the gelation of BSA. The addition of high-concentrated GSSG could substitute the formation of intramolecular disulfide bond and further restrain BSA gelation. As the occurrence of S-glutathionylation on approximate 9 pairs of intramolecular disulfide bonds, the secondary structure of BSA-SSG showed similarity as the original protein structure except that it has completely lost its chaperone-like activity. The same results could be reproduced in the case of gellable ovalbumin, emphasizing the formation of intramolecular disulfide bond induces the occurrence of gelation and chaperone-like activity on BSA and ovalbumin. Very few effective methods have been developed to quantify chaperone activity for various chaperones with different molecular weights and protection mechanisms. This last part of the thesis focused on studying the chaperone activity of BSA, ovalbumin and mini-αA-crystallin interacted with three target proteins including catalase, diamine oxidase and γ-globulin. It was found a high-positive correlation between log([chaperone]/[target protein]) and the percentage of turbidity variation (A%). The x-interception of trendline indicates the minimal concentration of the chaperone to manifest its efficiency, and the concentration is known as critical chaperone concentration (Ccp). Moreover, the slope of trendline, which demonstrates the reducible percentage of turbidity variation when the concentration of chaperone is 10 times over target protein, is able to represent the efficiency of chaperone activity on chaperone protein. The results indicated that ovalbumin has the least minimal Ccp to the three kinds of target protein. The efficiency of chaperone activity of mini-αA-crystallin on catalase, diamine oxidase and γ-globulin are 0.9392, 0.7032 and 1.1770, respectively, which are higher than those of BSA (0.4974, 0.4852 and 0.9330) and ovalbumin (0.6654, 0.3287 and 0.5145). These results indicate mini-αA-crystallin has preferable chaperone efficiency. This methodology provides an index on the compatibility of the efficiency on chaperone activiy.