Effects of papain digestion on the photocurrent responses of bacteriorhodopsin

• Main Authors: CHEN XIN YU, 陳欣禹
• Other Authors: Hsiu-Mei Chen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/35494853945154021272

碩士 === 國立臺灣科技大學 === 化學工程系 === 99 === In this study, limited papain-proteolysis of bacteriorhodopsin (BR) residing in the purple membrane (PM) of halophilic Halobacterium salinarum was employed to investigate the digestion effects on the photocurrents of drop-casted PM chips. The PM colors changed from purple to blue with the increase of digestion time, the same phenomenon also observed while the pH of PM suspension solutions was decreased from alkaline to acidic. The photocurrent polarity of native PM-coated chips in a pH 8.5 electrolyte changed upon 14-h prolonged digestion, which was also observed with an undigested native PM chip in acidic electrolytes. On the other hand, the photocurrent polarity of D96N PM-coated chips in a pH 8.5 electrolyte remained the same throughout the whole digestion process, implying the retardation of BR proton-uptake caused by Asp96→Asn mutation could not be completely overcome by the enhancement of the proton-uptake rate with papain digestion. Furthermore, the electrolyte pH where the photocurrent polarity of native PM-coated chips reversed increased with the increase of digestion time, while the value remained at pH 4.0 for the D96N ones, suggesting the effect of papain-digestion on the proton pump of BR. Moreover, the addition of NaN3 in the electrolyte also affected the proton pump and reversed the photocurrent polarity of the digested PM chips back to the original undigested state. The replacement of the counter electrode from blank ITO to ITO coated with undigested and 18-h digested PM decreased and increased the photocurrents, respectively, indicating the photocurrents of PM were addable. Finally, the labeling with 5-(iodoacetamido) fluorescein yielded an increase of absorbance at 495 nm for G241C PM, but not for the digested PM, confirming the removal of the Cys241-containing C-terminal peptide by papain digestion. Tuning the 495-nm absorbance values by changing the pH and salt concentrations of suspension solutions for the labeled G241C PM led to an estimate of its surface charge as -0.5 e- / BR.