pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.
A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.
Roy
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Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Gupta
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MN
D002918:Chymotrypsin / Q000378:metabolism*; D004146:Dioxanes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D011109:Polymethacrylic Acids / Q000378:metabolism*; D011489:Protein Denaturation; D020673:Protein Renaturation; D014508:Urea / Q000378:metabolism
D004146:Dioxanes; D011109:Polymethacrylic Acids; C038300:methylmethacrylate-methacrylic acid copolymer; D014508:Urea; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; C025223:1,4-dioxane