title: Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH).
abstract: The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.
authors.affiliation: Laboratoire de Génie Protéique et Cellulaire, Université de La Rochelle, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Trovaslet
authors.forename: Sandrine
authors.initials: S
authors.lastname: Dallet-Choisy
authors.forename: Filip
authors.initials: F
authors.lastname: Meersman
authors.forename: Karel
authors.initials: K
authors.lastname: Heremans
authors.forename: Claude
authors.initials: C
authors.lastname: Balny
authors.forename: Marie Dominique
authors.initials: MD
authors.lastname: Legoy
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D019281:Dimerization; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000737:chemistry
chemical_list: D014364:Tryptophan; D000426:Alcohol Dehydrogenase