Time-dependent structure and activity changes of alpha-chymotrypsin in water/alcohol mixed solvents.
Secondary structure of alpha-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. Alpha-chymotrypsin formed beta-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, alpha-chymotrypsin took on a native-like structure, which gradually changed to beta conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, alpha-chymotrypsin tended to form stable beta-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to alpha-helix structure with substantial loss in catalytic activity was observed when alpha-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, alpha-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of beta-sheet structure.
Sato
M
M
Institute of Materials Science, University of Tsukuba, Ibaraki, Japan.
Sasaki
T
T
Kobayashi
M
M
Kise
H
H
D000438:Alcohols / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D000431:Ethanol / Q000737:chemistry; D007700:Kinetics; D000432:Methanol / Q000737:chemistry; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water
D000438:Alcohols; D012997:Solvents; D014867:Water; D000431:Ethanol; D014270:Trifluoroethanol; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D000432:Methanol