The Effect of Dithiothreitol on Denaturation and Renaturation Procedure of Lysozyme
Date Issued
2007
Date
2007
Author(s)
Chang, Chi-Hsiung
DOI
zh-TW
Abstract
Observation of the conformational change of hen egg-white lysozyme during the denaturation procedure and quantitative analysis of oxidative kinetics of dithiothreitol (DTT) and refold lysozyme under different denaturation conditions via direct dilution method were investigated in this research. By size-exclusion chromatography (SEC), we were able to compare the apparent sizes of denatured lysozyme at different denaturation times with that of the native conformer. Furthermore, the measurements of activity and cysteine concentration also helped us to monitor the disruptions of active region and disulfide linkages of lysozyme during the course of denaturation and renaturation processes.
As shown in our results, the reaction of DTT oxidation at 25℃ and pH 8.2 followed the first order kinetics with a rate constant of 0.0994 day-1. In addition, we found that the rate of DTT oxidation was controlled by the diffusion rate of oxygen. That is, the magnitude of the rate constant of DTT oxidation was determined by the diffusion condition which is affected by the geometry of reaction vessel in use (e.g.: the height and cross-sectional area of tube) and the concentration of oxygen.
In the denaturation procedure, we found that, with insufficient DTT, the presence of urea would shift the peak of denatured lysozyme to the right (larger retention time), which means a more compact lysozyme conformer was formed resulted from the presence of urea after the complete depletion of DTT. According to our experimental results along with others found in the literature, we suggested that the first and the last reduced disulfide bond of lysozyme during the denaturation procedure were Cys76-Cys94 and Cys30-Cys115, respectively. Moreover, it appeared that the less the numbers of disulfide bond being reduced, the better the activity recovered in the renaturation procedure. Our results showed that the activity recovery of lysozyme would be obviously suppressed when the concentration of DTT carried over from the denaturing buffer to renaturation solution was higher than 0.8mM.
Subjects
溶菌酶
二硫代蘇糖醇
雙硫鍵
變性
復性
大小排阻層析法
lysozyme
dithiothreitol(DTT)
disulfide bonds
denaturation
renaturation
size-exclusion chromatography(SEC)
Type
thesis
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