Fluorescence and FTIR study of the pressure-induced denaturation of bovinepancreas trypsin

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 indic ated 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 st andard 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 differe nt 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 t ryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. interestingly, t he study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversi ble. FTIR spectroscopy was used to follow the changes in secondary structur e. The pressure stability data found by fluorescence measurements are confi rmed but the denaturation was irreversible at low and high pH in the FTIR i nvestigation. These findings confirm that the trypsin molecule has two doma ins: 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 stud y on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.