Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 5...

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Autores principales: Sutter, Marc, Oliveira, Sabrina, Sanders, Niek N., Lucas, Bart, van Hoek, Arie, Hink, Mark A., Visser, Antonie J. W. G., De Smedt, Stefaan C., Hennink, Wim E., Jiskoot, Wim
Formato: Texto
Lenguaje:English
Publicado: Kluwer Academic Publishers-Plenum Publishers 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1915606/
https://www.ncbi.nlm.nih.gov/pubmed/17294134
http://dx.doi.org/10.1007/s10895-007-0156-6
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author Sutter, Marc
Oliveira, Sabrina
Sanders, Niek N.
Lucas, Bart
van Hoek, Arie
Hink, Mark A.
Visser, Antonie J. W. G.
De Smedt, Stefaan C.
Hennink, Wim E.
Jiskoot, Wim
author_facet Sutter, Marc
Oliveira, Sabrina
Sanders, Niek N.
Lucas, Bart
van Hoek, Arie
Hink, Mark A.
Visser, Antonie J. W. G.
De Smedt, Stefaan C.
Hennink, Wim E.
Jiskoot, Wim
author_sort Sutter, Marc
collection PubMed
description The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ(max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.
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spelling pubmed-19156062007-07-13 Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red Sutter, Marc Oliveira, Sabrina Sanders, Niek N. Lucas, Bart van Hoek, Arie Hink, Mark A. Visser, Antonie J. W. G. De Smedt, Stefaan C. Hennink, Wim E. Jiskoot, Wim J Fluoresc Original Paper The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ(max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages. Kluwer Academic Publishers-Plenum Publishers 2007-02-09 2007-03 /pmc/articles/PMC1915606/ /pubmed/17294134 http://dx.doi.org/10.1007/s10895-007-0156-6 Text en © Springer Science+Business Media, LLC 2007
spellingShingle Original Paper
Sutter, Marc
Oliveira, Sabrina
Sanders, Niek N.
Lucas, Bart
van Hoek, Arie
Hink, Mark A.
Visser, Antonie J. W. G.
De Smedt, Stefaan C.
Hennink, Wim E.
Jiskoot, Wim
Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title_full Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title_fullStr Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title_full_unstemmed Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title_short Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
title_sort sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye nile red
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1915606/
https://www.ncbi.nlm.nih.gov/pubmed/17294134
http://dx.doi.org/10.1007/s10895-007-0156-6
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