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Fluorescence monitoring of trypsin adsorption in layer-by-layer membrane systems

Paper ID Volume ID Publish Year Pages File Format Full-Text
17185 42650 2012 9 PDF Available
Title
Fluorescence monitoring of trypsin adsorption in layer-by-layer membrane systems
Abstract

A combined fluorescence analysis, involving the use of steady-state fluorescence and fluorescence anisotropy was used, allowing eliciting information about the structural changes induced on trypsin after exposure to membrane surfaces with diverse chemistry, designed through a layer-by-layer methodology. Using this monitoring strategy it was possible to understand the influence of the surface chemistry on the structural characteristics of the attached proteins and how they relate to changes of their activity resulting from the adsorption process. This knowledge may be used to direct the development of surfaces with suitable chemistry, leading enzymatic-based processes with improved performance. The results obtained show clearly that trypsin exposed to different membrane surfaces, changes its conformation, either if it adsorbs to the membrane or if it remains in solution. A significant loss of enzymatic activity was observed upon the adsorption process, for the adsorbed and non-adsorbed protein. This loss of the trypsin activity was correlated with the presence of molecular unfolding events that mediate trypsin–membrane surface interactions and the decrease of the molecular mobility of the adsorbed trypsin, which was shown to be dependent on the chemical characteristics of the membrane surface. Changes on the selectivity of the adsorbed trypsin were also observed, and may be ruled by the strength of the enzyme–surface interactions established.

► Trypsin contact to layer-by-layer based AN69 membranes promotes protein unfolding. ► Enzymatic activity losses upon contact to the surface relate with trypsin unfolding. ► Fluorescence monitors distinct trypsin flexibilities when adsorbed at AN69 surfaces. ► The trypsin molecular flexibility depends on the enzyme–surface interaction strength. ► Selectivity of the adsorbed trypsin depends on its structural flexibility.

Keywords
Fluorescence; Trypsin; Protein adsorption; Protein–membrane interactions; Enzymatic activity; Layer-by-layer membranes
First Page Preview
Fluorescence monitoring of trypsin adsorption in layer-by-layer membrane systems
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issues 6–7, 10 December 2012, Pages 325–333
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering