title: 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.
authors.affiliation: Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XL, UK.
authors.forename: G A
authors.initials: GA
authors.lastname: Hutcheon
authors.forename: M C
authors.initials: MC
authors.lastname: Parker
authors.forename: B D
authors.initials: BD
authors.lastname: Moore
mesh_terms: D000388:Air; D001407:Bacillus / Q000201:enzymology; D001709:Biotechnology / Q000379:methods*; D003903:Deuterium; D004798:Enzymes / Q000737:chemistry*; D005612:Freeze Drying; D006586:Hexanes; D006859:Hydrogen; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D012997:Solvents; D013381:Subtilisins / Q000302:isolation & purification
chemical_list: D004798:Enzymes; D006586:Hexanes; D012997:Solvents; C026385:n-hexane; D006859:Hydrogen; D003903:Deuterium; D013381:Subtilisins