EPR Spectra of Spin-labeled Fatty Acids in Detergent Solutions: “Immobilized Species” in the...
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Transcript of EPR Spectra of Spin-labeled Fatty Acids in Detergent Solutions: “Immobilized Species” in the...
EPR Spectra of Spin-labeled Fatty Acids in DetergentSolutions: ‘‘Immobilized Species’’ in the Absenceof Protein
Stefan Kuntz • Fabian Leinisch • Kalman Hideg •
Wolfgang E. Trommer
Received: 2 June 2009 / Published online: 19 November 2009
� Springer 2009
Abstract Electron paramagnetic resonance spectra of 5-doxyl-stearic acid, 3-(2-
Dodecyl-1-oxyl-5,5-dimethylpyrrolidin-2-yl)propanoic acid radical (4-proxyl-pal-
mitic acid) and of 3-(1-Oxyl-2,5-dimethyl-5-dodecylpyrrolidine-2-yl)propanoic acid
radical (4-azetoxyl-stearic acid) in lipid micelles of nonaethyleneglycol-mono-
dodecyl ether (C12E9) and pentaethyleneglycol-monooctyl ether (C8E5) can reveal
immobilized components typical of spin-labeled protein-bound fatty acids as well as
phospholipids. The occurrence of such species depends on the detergent, the relative
orientation of the nitroxide group with respect to the longitudinal axis and the
molecular structure of the fatty acid as well as on polarity and ionic strength of the
aqueous phase. Whereas 5-doxyl-stearic acid and 4-proxyl-palmitic acid exhibited
these highly immobilized components in C12E9 micelles but not in C8E5, only a
single relatively mobile component was observed with 4-azetoxyl-stearic acid in
both detergents. The immobilized component is lost by the addition of isopropanol
but not as much by ethanol and also when distilled water is substituted for the
buffer.
1 Introduction
Binding of annular lipids and fatty acids to membrane proteins has often been
followed by means of spin-labeled derivatives. For an early review see Ref. [1]. The
corresponding electron paramagnetic resonance (EPR) spectra are then typical for
S. Kuntz � F. Leinisch � W. E. Trommer (&)
Department of Chemistry, Kaiserslautern University of Technology, Postfach 3049,
67653 Kaiserslautern, Germany
e-mail: [email protected]
K. Hideg
Institute of Organic and Medicinal Chemistry, University of Pecs, Pecs, Hungary
123
Appl Magn Reson (2010) 37:455–461
DOI 10.1007/s00723-009-0057-8
Applied
Magnetic Resonance
one or even several highly immobilized components with the hyperfine structure
2Azz values of 45 G and more in a hydrophobic environment [2]. However, spin-
labeled lipids and even spin-labeled fatty acids may exhibit EPR spectra similar if
not identical to those in the presence of proteins to which they may be bound and
hence, leading to misinterpretation of data obtained in the presence of the protein.
Here we describe the EPR spectra of 5-doxyl-stearic acid and of 3-(1-oxyl-2,
5-dimethyl-5-dodecylpyrrolidine-2-yl)propanoic acid radical (4-azetoxyl-stearic
acid) as well as of 3-(2-dodecyl-1-oxyl-5,5-dimethylpyrrolidin-2-yl)propanoic acid
radical (4-proxyl-palmitic acid) (Fig. 1) under various conditions in nonaethyle-
neglycol-monododecyl ether (C12E9) and pentaethyleneglycol-monooctyl ether
(C8E5) (Fig. 2) micelles in the absence of any proteins.
2 Materials and Methods
4-Proxyl-palmitic acid and 4-azetoxyl-stearic acid were synthesized as previously
described in [3]. 5-Doxyl-stearic acid, nonaethyleneglycol-monododecyl ether
and pentaethyleneglycol-monooctyl ether were purchased from Sigma. All other
chemicals were of a reagent grade from various sources.
Fig. 1 Structural formulasof 5-doxyl-palmitic acid (a),4-azetoxyl-stearic acid (b) andof 4-proxyl-palmitic acid (c)
aCH3(CH2)11(OCH2CH2)9OH
bCH3(CH2)7(OCH2CH2)5OH
Fig. 2 Structural formulas of nonaethyleneglycol-monododecyl ether (C12E9) (a) and pentaethyleneglycol-monooctyl ether (C8E5) (b)
456 S. Kuntz et al.
123
3 EPR Spectroscopy
EPR spectra were measured on a Bruker Elexsys E580 EPR spectrometer operating in
the X-band mode (9.7 GHz). A dielectric cavity was used for all the experiments. EPR
spectra were recorded at 297 K with a scan width of 100 G and a center field of
3452 G. The microwave power was set to 8 mW, and the receiver gain to 72 dB at a
modulation amplitude of 1 G. The baseline was subtracted from the recorded spectra.
4 Results
4.1 EPR Spectra of 4-Proxyl-Palmitic Acid and 5-Doxyl-Stearic Acid
in the Presence of C12E9 and C8E5 Micelles
Figure 3a shows the EPR spectrum of 4-proxyl-palmitic acid in micelles of
nonaethyleneglycol-monododecyl ether (C12E9). The spectrum exhibits two distinct
peaks each in the low-field as well as the high-field region as indicated by arrows.
Besides a weakly immobilized component denoted 1, a second strongly immobi-
lized component 2 with an Azz value of 46.5 G is clearly visible. In contrast, the
spectrum of the same compound, 4-proxyl-palmitic acid, in the presence of
pentaethyleneglycol-monooctyl ether (C8E5) shows one component, only (Fig. 3b)
typical of weak immobilization. Hence, switching from C12E9 to C8E5 has a strong
effect on the resulting EPR spectra.
Fig. 3 EPR spectra of 100 lM4-proxyl-palmitic acid in thepresence of C12E9 and C8E5
micelles contained in 0.1 MTris–HCl, 2 mM Tris–EDTA,pH 7.2, at 24 �C. a 1% C12E9
and b 1% C8E5
EPR Spectra of Spin-Labeled Fatty Acids in Detergents 457
123
Corresponding measurements with 5-doxyl-stearic acid yielded very similar
results as shown in Fig. 4a, b. Again, two components were observed with C12E9 as
detergent (Fig. 4a), whereas in C8E5 only a weakly immobilized species is seen
(Fig. 4b). Although the concentration of 5-doxyl-stearic acid was the same as that of
the proxyl derivative (100 lM), the signal intensities were considerably lower. As
has been found out later, the nitroxide group of the analog was partially reduced.
However, this should not have affected our results.
4.2 EPR Spectra of 4-Azetoxyl-Stearic Acid in the Presence of C12E9 and C8E5
Micelles
The nitroxide group of 4-azetoxyl-stearic acid is oriented perpendicular to the long
axis of the fatty acid as can be seen in Fig. 1. EPR spectra of this analog did not
show the immobilized component even in the presence of C12E9 (Fig. 5) as was
observed for 4-proxyl-palmitic and 5-doxyl-stearic acid. C8E5 yielded a virtually
identical spectrum (data not shown).
4.3 Effects of Organic Solvent and Ionic Strength on the EPR Spectra
of 4-Proxyl-Palmitic Acid in the Presence of C12E9 Micelles
Since fatty acids and, certainly their spin-labeled derivatives as well, are barely
soluble in water, stock solutions are often prepared in organic solvents and aliquots
Fig. 4 EPR spectra of 200 lM5-doxyl-stearic acid in thepresence of C12E9 and C8E5
micelles contained in 0.1 MTris–HCl, 2 mM Tris–EDTA,pH 7.2, at 24 �C. a 1% C12E9
and b 1% C8E5
458 S. Kuntz et al.
123
added to the aqueous biological system. However, the resulting EPR spectra can
vary dramatically with the final concentration of the organic solvent. Figure 6 shows
the effect of isopropanol on the EPR spectra of 4-proxyl-palmitic acid in C12E9 with
the immobilized component completely disappearing at 16% of the organic solvent.
In contrast, ethanol had virtually no effect on the spectral line shape up to 25%.
Very similar effects were observed in relation to the ionic strength of the aqueous
phase (Fig. 7). In distilled water the immobilized component is almost completely
lost with but a slight broadening of the high-field minimum. Upon going from 1 M
Fig. 5 EPR spectrum of100 lM 4-azetoxyl-stearic acidin the presence of 1% C12E9
micelles contained in 0.1 MTris–HCl, 2 mM Tris–EDTA,pH 7.2, at 24 �C
Fig. 6 EPR spectra of 100 lM4-proxyl-palmitic acid in thepresence of 1% C12E9 micellescontained in 0.1 M Tris–HCl,2 mM Tris–EDTA, pH 7.2, at24 �C. a In the presence ofincreasing concentrations ofisopropanol and b of ethanol
EPR Spectra of Spin-Labeled Fatty Acids in Detergents 459
123
to 100 and 10 mM Tris–HCl, the ratio between the low-field amplitudes gradually
shifts towards the more mobile component (Fig. 7).
5 Discussion
This study does not claim to provide novel insights in the understanding of EPR
spectra of spin-labeled fatty acids in membranes or detergents. However, it is shown
that various experimental parameters may easily lead to erroneous interpretation of
the data, and that the kind of even closely related detergents may play a significant
role. Most of the early studies focused on ordered lipid multilayers and/or the
angular dependence of the resulting EPR spectra [4, 5]. Indeed, spectral differences
and even two-component systems in liposomes composed of natural lipid mixtures
have been observed in the absence of proteins depending on the position of the spin
label along the fatty acid chain [6]. Doxyl-fatty acids with the spin label at position
five exhibited two components, whereas with the12-doxyl derivate a shoulder was
observed and a single component in case of 16-doxyl. Clearly, the far more
restricted motion near the polar head group allows for detection of the parallel and
perpendicular components of the hyperfine structure A-tensor, Ak and A\ as was
nicely shown for 5-doxyl-tricosanoic acid [7]. Due to the increasing flexibility the
motional differences and the resulting anisotropy are averaged out further down the
chain.
To the best of our knowledge, here we report for the first time significant
detergent-dependent spectral differences in the absence of orientation-dependent
anisotropy. Clearly, larger liposomes exhibit reduced rotational correlation times and
hence, larger 2 Azz values of the incorporated spin-labeled fatty acids. The size of
micelles composed of ethyleneglycol-derived detergents depends on the length of
their apolar moiety [8]. The EPR spectra, however, also reveal that not only the
overall tumbling rate is reduced, but a restricted motion of the spin-labeled fatty
acids in C12E9 and thus, allowing for distinct parallel and perpendicular components.
This is seen only with the 4-proxyl- and 5-doxyl fatty acids, not with 4-azetoxyl-
stearic acid. In the latter, the nitroxide group is oriented perpendicular to the long
Fig. 7 EPR spectra of 100 lM4-proxyl-palmitic acid in thepresence of 1% C12E9 micellesat 24 �C contained in 2 mMTris–EDTA, pH 7.2, at 24 �C atdifferent concentrations of Tris–HCl
460 S. Kuntz et al.
123
axis and not parallel as in the former. The reason for the almost complete averaging
of the anisotropic components of the hyperfine interaction most likely resides in the
molecular geometry with the nitroxide moiety fully integrated in the elongated
molecule. All these effects, however, are not observed in the smaller C8E5 micelles.
The addition of organic solvents allows the detergent as well as the spin-labeled
fatty acids to dissolve in the ‘‘aqueous phase’’ and thus, reducing the motional
restriction. In this respect, isopropanol is more efficient than ethanol (Fig. 6).
Likewise, a higher salt concentration stabilizes separate aqueous and detergent
phases (Fig. 7).
In conclusion, binding studies of spin-labeled fatty acids and lipids to membrane
proteins solubilized in detergents require careful controls in the absence of protein.
References
1. O.H. Griffith, P.C. Jost, L.J. Berliner (eds.), Spin Labeling: Theory and Applications (Academic Press,
New York, 1976), pp. 454–523
2. P. Jezek, M. Bauer, W.E. Trommer, FEBS. Lett. 361, 303–307 (1995)
3. M. Balog, C. Abe, T. Kalai, H.-J. Steinhoff, J. Jeko, K. Hideg, Synthesis. 1663–1670 (2007)
4. I.C.P. Smith, K.W. Butler, L.J. Berliner (eds.), Spin Labeling: Theory and Applications (Academic
Press, New York, 1976), pp. 411–451
5. D. Marsh, G. Cevec, Phospholipid Bilayers, Physical Principles and Models (Wiley, Chichester, 1987)
6. P.J. Dehlinger, P.C. Jost, O.H. Griffith, Proc. Natl. Acad. Sci. USA. 71, 2280–2284 (1974)
7. W.L. Hubbell, H.M. McConnell, Proc. Natl. Acad. Sci. USA. 64, 20–27 (1969)
8. A. Helenius, D. McCaslin, E. Fries, C. Tanford, Methods Enzymol. 56, 734–749 (1979)
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