Measuring enzyme motility in organic media using novel H-D exchange methodology.

Abstract:

: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.

journal_name

Biotechnol Bioeng

authors

Hutcheon GA,Parker MC,Moore BD

subject

Has Abstract

pub_date

2000-11-05 00:00:00

pages

262-9

issue

3

eissn

0006-3592

issn

1097-0290

pii

10.1002/1097-0290(20001105)70:3<262::AID-BIT3>3.0.

journal_volume

70

pub_type

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