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Measuring enzyme motility in organic media using novel H‐D exchange methodology
Author(s) -
Hutcheon Gillian A.,
Parker Marie Claire,
Moore Barry D.
Publication year - 2000
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 - 10.1002/1097-0290(20001105)70:3<262::aid-bit3>3.0.co;2-6
Subject(s) - subtilisin , chemistry , hexane , aqueous solution , solvent , dissolution , hydrogen–deuterium exchange , chromatography , nuclear chemistry , enzyme , hydrogen , organic chemistry
A novel deuterium ( 2 H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen‐deuterium (H‐D) exchange. Using this methodology differences in the H‐D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity ( a w ), the degree of H‐D exchange increased rapidly with hydration (from anhydrous to a w 0.22). At a w 0.22, subtilisin powders hydrated in air were found to have reached an H‐D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a w ) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n ‐hexane showed a significant reduction in H‐D exchange at all a w studied. Control experiments demonstrated that the reduction in H‐D exchange observed for subtilisin in n ‐hexane was not a kinetic effect. This lower level of exchange in n ‐hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 262–269, 2000.