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

Biotechnol Bioeng. 2000 Nov 5;70(3):262-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Air
  • Bacillus / enzymology
  • Biotechnology / methods*
  • Deuterium
  • Enzymes / chemistry*
  • Freeze Drying
  • Hexanes
  • Hydrogen
  • In Vitro Techniques
  • Magnetic Resonance Spectroscopy
  • Protein Conformation
  • Solvents
  • Subtilisins / isolation & purification

Substances

  • Enzymes
  • Hexanes
  • Solvents
  • n-hexane
  • Hydrogen
  • Deuterium
  • Subtilisins