Open AccessFluorescence and FTIR study of the pressure‐induced denaturation of bovine pancreas trypsin
Author(s) -
Ruan Kangcheng,
Lange Reinhard,
Meersman Filip,
Heremans Karel,
Balny Claude
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00665.x
Subject(s) - denaturation (fissile materials) , chemistry , tryptophan , trypsin , fourier transform infrared spectroscopy , fluorescence spectroscopy , fluorescence , protein secondary structure , bovine serum albumin , chromatography , analytical chemistry (journal) , biochemistry , enzyme , nuclear chemistry , amino acid , quantum mechanics , physics
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 °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.