Abstract: The conformational changes of native trypsin and DHPM-induced unfolded trypsin during the denaturation by urea have been investigated using the methods of ultraviolet (UV) spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Results indicated that native trypsin showed the most significant changes when the concentration of urea was from 4.0 to 6.0 mol/L. The absorbance at 280 nm decreased from 0.2746 to 0.1824, and the absorption peak showed red shift from 279 to 283 nm; the relative fluorescence intensity decreased from 105.9 to 98.27, and the emission peak showed red shift from 352 to 355 nm. The percentage of β-sheet content decreased from 46.9% to 30.6%. In contrast, the most significant changes were observed in unfolded trypsin when the concentration of urea was from 2.0 to 4.0 mol/L. The UV absorbance decreased from 0.3121 to 0.2159 and the peak showed red shift from 281 to 283 nm; the fluorescence intensity decreased from 108.24 to 99.07 and the emission peak showed red shift from 353 to 360 nm. The percentage of β-sheet content decreased from 40.6% to 38.9%. These findings show that urea has a more significant impact on DHPM-induced unfolded trypsin compared with native trypsin. In the presence of urea at different concentrations, trypsin has different conformations of intermediates that show characteristics of molten globule state.

Key words: urea, trypsin, dynamic high-pressure microfluidization (DHPM), conformation