Refolding of partially and fully denatured lysozymes.
Lysozyme refolding with high yields sometimes results from incomplete denaturation. Dithiothreitol (DTT) is a reductant commonly used to reduce and unfold disulfide-stabilized lysozymes. Through the use of fluorescence spectroscopy to access the extent of denaturation, we found that the rate and extent of denaturation highly depended on the concentration of DTT. Further, the denaturation exhibited a two-phase transition at a high DTT concentration with DTT at >100 mM and long denaturation time (>24 h) being needed for complete denaturation. A low DTT concentration and a short denaturation time resulted in fast refolding with high activity recovery, while a high DTT concentration and a long denaturation time resulted in slow refolding with low activity recovery. Hence, the renaturation of disulfide-containing lysozyme was highly affected by the extent of denaturation.
Lin
Jiun-Liang
JL
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan.
Ruaan
Ruoh-Chyu
RC
Hsieh
Hsyue-Jen
HJ
D000818:Animals; D002645:Chickens; D004229:Dithiothreitol / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D014508:Urea; D009113:Muramidase; D004229:Dithiothreitol; D019803:Glutathione Disulfide