title: Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.
abstract: The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.
authors.affiliation: Shanghai Institute of Biochemistry, Academia Sinica, China.
authors.forename: K
authors.initials: K
authors.lastname: Ruan
authors.forename: R
authors.initials: R
authors.lastname: Lange
authors.forename: F
authors.initials: F
authors.lastname: Meersman
authors.forename: K
authors.initials: K
authors.lastname: Heremans
authors.forename: C
authors.initials: C
authors.lastname: Balny
mesh_terms: D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D010179:Pancreas / Q000201:enzymology; D011312:Pressure; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
chemical_list: D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D014508:Urea; D014357:Trypsin