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

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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.
Original languageUnknown
Pages (from-to)325-333
JournalEnzyme And Microbial Technology
Volume51
Issue numberNA
DOIs
Publication statusPublished - 1 Jan 2012

Cite this

@article{f693bf48afce414a96d03f24588b7b4b,
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.",
author = "Portugal, {Carla Alexandra Moreira} and Crespo, {Jo{\~a}o Paulo Serejo Goul{\~a}o}",
year = "2012",
month = "1",
day = "1",
doi = "10.1016/j.enzmictec.2012.07.016",
language = "Unknown",
volume = "51",
pages = "325--333",
journal = "Enzyme And Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier Science B.V., Amsterdam.",
number = "NA",

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TY - JOUR

T1 - Fluorescence monitoring of trypsin adsorption in layer-by-layer membrane systems

AU - Portugal, Carla Alexandra Moreira

AU - Crespo, João Paulo Serejo Goulão

PY - 2012/1/1

Y1 - 2012/1/1

N2 - 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.

AB - 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.

U2 - 10.1016/j.enzmictec.2012.07.016

DO - 10.1016/j.enzmictec.2012.07.016

M3 - Article

VL - 51

SP - 325

EP - 333

JO - Enzyme And Microbial Technology

JF - Enzyme And Microbial Technology

SN - 0141-0229

IS - NA

ER -