Abstract
The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 μM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 μM-1 and for UV excitation at 295 nm was 3.2 μM-1. In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.
| Original language | English |
|---|---|
| Pages (from-to) | 19-23 |
| Number of pages | 5 |
| Journal | Journal of Photochemistry and Photobiology B: Biology |
| Volume | 92 |
| Issue number | 1 |
| DOIs | |
| State | Published - 24 Jul 2008 |
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Keywords
- 8-Anilino-1-naphthalene sulfonate
- Fluorescence anisotropy
- Fluorescence lifetime
- Lecithin cholesterol acyltransferase
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Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques. / Sarkar, Pabak; Bharill, Shashank; Gryczynski, Ignacy; Gryczynski, Zygmunt; Nair, Maya P.; Lacko, Andras G.
In: Journal of Photochemistry and Photobiology B: Biology, Vol. 92, No. 1, 24.07.2008, p. 19-23.Research output: Contribution to journal › Article
TY - JOUR
T1 - Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques
AU - Sarkar, Pabak
AU - Bharill, Shashank
AU - Gryczynski, Ignacy
AU - Gryczynski, Zygmunt
AU - Nair, Maya P.
AU - Lacko, Andras G.
PY - 2008/7/24
Y1 - 2008/7/24
N2 - The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 μM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 μM-1 and for UV excitation at 295 nm was 3.2 μM-1. In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.
AB - The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 μM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 μM-1 and for UV excitation at 295 nm was 3.2 μM-1. In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.
KW - 8-Anilino-1-naphthalene sulfonate
KW - Fluorescence anisotropy
KW - Fluorescence lifetime
KW - Lecithin cholesterol acyltransferase
UR - http://www.scopus.com/inward/record.url?scp=46749083458&partnerID=8YFLogxK
U2 - 10.1016/j.jphotobiol.2008.03.009
DO - 10.1016/j.jphotobiol.2008.03.009
M3 - Article
C2 - 18485727
AN - SCOPUS:46749083458
VL - 92
SP - 19
EP - 23
JO - Journal of Photochemistry and Photobiology, B: Biology
JF - Journal of Photochemistry and Photobiology, B: Biology
SN - 1011-1344
IS - 1
ER -