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 beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (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 beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-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 beta-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.
Sutter
Marc
M
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands. m.sutter@lacdr.leidenuniv.nl
Oliveira
Sabrina
S
Sanders
Niek N
NN
Lucas
Bart
B
van Hoek
Arie
A
Hink
Mark A
MA
Visser
Antonie J W G
AJ
De Smedt
Stefaan C
SC
Hennink
Wim E
WE
Jiskoot
Wim
W
D002850:Chromatography, Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000032:analysis*; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D001616:beta-Galactosidase / Q000032:analysis
D005456:Fluorescent Dyes; D010078:Oxazines; D011506:Proteins; D012996:Solutions; D001616:beta-Galactosidase; C044808:nile red