Structural and functional characterization of solution, gel, and aggregated forms of trypsin in organic solvent-assisted and pH-induced phase changes

Abstract

In this study the effect of three different physicochemical parameters on pHtriggered gelation and aggregation of bovine pancreatic trypsin changes and structural and functional changes in these changes in alcohol-water mixtures were studied. Methods: Trypsin gelation times were studied using inverted tube method. Trypsin stability was studied using trypsin enzyme assay. Protein secondary structural changes were monitored using FTIR spectroscopy. Gel and aggregate macrostructures and morphologies were viewed using Scanning Electron Microscopy. Results: The solution phase was observed in the absence of both NaOH and CaCl2. The gel phase was observed in the absence of the either. The aggregate phase was observed in the presence of the both agents all depending on trypsin concentrations used. Trypsin stability studies showed that there were a nearly 53 and 32% specific activity losses after the gelation and aggregation processes. According to FTIR studies β–sheet structure in 1637 cm-1 band disappeared in trypsin gel and trypsin aggregates. Increases in α–helix structure in 1651 cm-1 in trypsin gel and aggregates were observed. Iodoacetamide delayed the gelation and prevented the aggregation indicating the importance of intermolecular disulfides in the both processes. Conclusion: Trypsin gelation was caused by the denaturation of the protein three dimensional structures. The gel and aggregate formation indicates a secondary structural change towards α–helix structure formation at the expense of β–sheet structure and formation of intermolecular disulfide bonds.

Amaç: Bu çalışmada alkol-su karışımlarında pH tarafından tetiklenen sığır pankreatik tripsin jelleşmesi ve agregasyonu faz değişiklikleri üzerine üç değişik fizikokimyasal değişkenin yapısal ve fonksiyonel etkileri çalışılmıştır. Metod: Tripsin jelleşmesi ters tüp metodu ile çalışılmıştır. Tripsin stabilitesi tripsin enzim aktivitesi tayiniyle çalışılmıştır. Protein ikinci yapı değişiklikleri FTIR spektroskopisi metodu ile gözlenmiştir. Jel ve agregat makroyapıları ve morfolojileri taramalı elektron mikroskopisi yöntemiyle incelenmiştir.