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Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?
Author(s) -
Griebenow Kai,
Klibanov Alexander M.
Publication year - 1997
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19970220)53:4<351::aid-bit1>3.0.co;2-m
Subject(s) - subtilisin , chemistry , dimethyl sulfoxide , protein secondary structure , solvent , aqueous solution , solubility , denaturation (fissile materials) , dissolution , organic chemistry , fourier transform infrared spectroscopy , catalysis , circular dichroism , enzyme , chromatography , stereochemistry , nuclear chemistry , biochemistry , chemical engineering , engineering
We developed an FTIR (Fourier transform infrared) methodology for quantitatively assessing the secondary structure of proteins suspended in nonaqueous media. This methodology was used to measure the percentages of α‐helices and β‐sheets of subtilisin Carlsberg, prepared under different conditions, placed in various organic solvents. The title question was addressed with respect to some instances of markedly influencing the subtilisin activity in organic solvents reported in the literature. It is concluded that the mechanism of subtilisin activation by KCl and N ‐Ac‐L‐Phe‐NH 2 present in the aqueous solution of the enzyme prior to lyophilization may be due to their preservation of the secondary structure, otherwise altered by the dehydration. Likewise, subtilisin inactivation in the protein‐dissolving solvent DMSO (dimethyl sulfoxide) is likely caused by enzyme denaturation (the loss of both α‐helices and β‐sheets). On the other hand, some other ligands, as well as protein nondissolving organic solvents, while greatly affecting the subtilisin activity, have little effect on its secondary structure, thus ruling out the causal relationship between the two. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 351–362, 1997.