Proc. Natl. Acad. Sci. USA
Vol. 74, No. 5, pp. 1807-1810, May 1977
Biochemistry
Lipid-protein interaction in the phosphatidylcholine exchange protein (spin-labeled lecithin/electron spin resonance spectroscopy)
PHILIPPE F. DEVAUX*, PETER MOONENt, ALAIN BIENVENUE*, AND KAREL W. A. WIRTZt *
I.B.P.C., 13 rue Pierre et Marie Curie, 75005 Paris, France; and t Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, Padualaan,
Utrecht, The Netherlands
Communicated by Harden M. McConnell, January 10, 1977
MATERIALS AND METHODS Materials. The phosphatidylcholine exchange protein was purified from bovine liver according to established procedures (11). It was stored at -10° in 50% (vol/vol) glycerol at a concentration of approximately 100 gg (4.5 nmol) of protein per ml. Before use, the protein was dialyzed overnight against 20% (wt/vol) sucrose/20 mM sodium acetate/10 mM Tris-HCl, pH 6.6. Protein content was estimated by measuring absorbance at 280 nm (A280 = 0.100 is equivalent to 2.7 nmol of protein per ml). Spin-labeled analogs of lecithin (I and II) were synthesized according to Hubbell and McConnell (12). Their general formula can be written as:
ABSTRACT Incorporation of 2-acyl spin-labeled lecithin into the phosphatidylcholine exchange protein from bovine liver results in an immobilization of the spin-label at the methyl and the carboxyl terminal end of the acyl chain. The nitroxide group on the protein-bound lecithin molecule is not accessible to ascorbate. This suggests that lecithin is buried in a pocket on the protein, which effectively shields the acyl chains from the medium.
The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of spin-labeled lecithin from single bilayer vesicles to rat liver mitochondria (1). Because the protein acts as a carrier, a spin-labeled lecithin is bound to the protein upon removal from the membrane (2). Only those spin-labeled lec-
0 o
CH3-(CH2)mC-(CH2), 0
1
1
CH2-0-C-(CH2)14 -CH3
C O CH
N-0
0
1
CH2-(--P-O-(CH2)2-N(CH3)3 I: m,n = 1,14 LI: mn = 10,3
Phosphatidic acid was prepared from egg yolk lecithin by degradation with phospholipase D according to Davidson and Long (13) and stored as the sodium salt in chloroform at -20°. Vesicles containing the spin-labeled analogs and phosphatidic acid were prepared in sucrose/acetate/Tris buffer as previously described (14). Preparation of Samples. Exchange protein (final concentration, 2-3 AM) was mixed with spin-labeled lecithin vesicles (final concentration, 50-100 ,uM in lipids) containing 2 mol % phosphatidic acid. ESR measurements were performed directly on this mixture and on control samples without exchange pro-
ithin molecules that contained the spin-label on the acyl chain were transferred. Lecithin with the nitroxide in the polar head group was not transferred. This agrees with the notion that the protein contains a binding site that interacts specifically with the lecithin head group exposed at the membrane interface (ref. 3; H. H. Kamp et al., unpublished data). It has previously been demonstrated that the protein binds one molecule of lecithin that cannot be hydrolyzed by phospholipases (4). This indicated that the lecithin molecule was well embedded in the protein. The latter aspect has been elaborated in the present study in which the interaction between the spin-labeled 2-acyl chain of lecithin and the protein has been studied with electron spin resonance (ESR) spectroscopy. It will be shown that measurements of the mobility and accessibility of the nitroxide provide more direct information on the localization of the lecithin molecule in the exchange protein. Similar studies have been performed on the interaction of spin-labeled fatty acids with bovine serum albumin (5). Lipid-protein interactions in membranes have also been studied with spinlabeled lipids (6-10). In general, the latter studies have been complicated by the partitioning of spin-labels between protein and bilayers as well as by nonspecific binding. These problems do not arise when spin-labeled lecithin is bound to the soluble exchange protein.
tein. In some experiments, ESR measurements were performed on the spin-labeled lecithin bound to the protein in the absence of spin-labeled lecithin vesicles. After incubation in sucrose/ acetate/Tris buffer, protein and spin-labeled lecithin vesicles were separated by DEAE-cellulose chromatography. The vesicles that contained 7 mol % phosphatidic acid were retained by the DEAE-cellulose whereas the protein was eluted in the void volume. A chemical reduction of the protein-bound spin-labeled lecithin was attempted at 00 in the presence of 2 mM sodium
ascorbate. Electron Spin Resonance Spectra. ESR measurements were performed on a Varian E 109 spectrometer in 50- or 200-Iu quartz cells. The 200-,ul cell was used for measurements at room
Abbreviation: ESR, electron spin resonance.
1807
1808
Biochemistry: Devaux et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
I
F
0
FIG. 1. Spectra of spin-labeled lecithin I and II at room temperature. The distance between two vertical lines repesents 10 gausses (1 X 10-4 T). Left. The top spectrum corresponds to the accumulated spectra obtained with vesicles of spin label I (nitroxide near the methyl end) in the presence of exchange protein; the middle spectrum is the control in the absence of protein. The mixture contained 500 MM lecithin and 1-2 AM protein; the accumulation period was 3 hr. The lower spectrum is the difference (approximately 10-fold magnification). Right. Same as Left, except for the use of spin label II (nitroxide near the carboxyl end).
temperature and the 50-Al cell for measurements at controlled temperatures. The ESR spectrometer was connected to an In-
tertechnique Didac 4000 multichannel analyzer for data accumulation. Two- to 4-min scans were used, and the accumulation periods were 1-4 hr. These procedures were necessary because of the low protein concentration. The spectra of the spin-labeled lecithin vesicles recorded in the presence and absence of exchange protein were electronically subtracted to obtain the spectrum of the spin-labeled lecithin bound to the exchange protein. RESULTS AND DISCUSSION ESR spectra of spin-labeled lecithin bound to exchange protein The ESR spectrum of vesicles prepared from pure spin-labeled lecithin I or II is a single broad line. This reflects the spin-spin interactions of the lecithin molecules in the bilayer. Upon addition of exchange protein at room temperature, additional structures could be seen superimposed on this broad line. Because the protein concentration is in the order of 2-3 MM, the signal-to-noise ratio of a direct recording is poor. The spectra obtained after data accumulation at room temperature with pure spin-labeled vesicles I and II are shown in Fig. 1. From the direct recording it was apparent that, at room temperature or above, the spectrum characteristic for the exchange proteinvesicle mixture remained constant after 5 min of incubation. On the other hand, when proteins and vesicles were mixed and kept at 00, the superimposed structures became visible only after 1-2 hr. Mixing of protein and vesicles at room temperature followed by recording of the spectra at 00 produced the superimposed lines immediately. These experiments indicate that
the equilibrium governing the interaction of the protein with the spin-labeled vesicles is reached rapidly at room temperature and slowly at 00. The dissociation constant of exchange protein interacting with egg lecithin vesicles that contain 2 mol % phosphatidic acid is 6 X 10-3 M (15). Because the rate of transfer of spin-labeled lecithin I and II was of the same magnitude as that of egg lecithin (1), we assume that the dissociation constant of the exchange protein-spin labeled vesicle complex is of the same order. Given the high value of this constant it can be calculated that virtually no exchange protein is bound to the vesicles. This implies that the difference spectra presented in Fig. 1 represent the unbound protein containing spin-labeled lecithin. In order to obtain direct evidence for this conclusion, exchange protein was mixed with spin-labeled vesicles at room temperature and subsequently separated from these vesicles by DEAE-cellulose chromatography. ESR spectra of the protein present in the eluent were similar to the difference spectra presented in Fig. 1. Because of a more than 50% loss of protein as a result of chromatography, the signal-to-noise ratio of the accumulated spectra was low. A base-line subtraction was necessary. Therefore, in addition to being simpler, the method based on difference spectra gave better results. On the basis of previous studies (ref. 4; H. H. Kamp et al., unpublished data) the unbound protein molecule probably contains one molecule of spin-labeled lecithin after mixing with the spin-labeled vesicles. It is seen from Fig. 1 that the spectrum of the protein-bound spin-labeled lecithin has a similar overall shape independent of whether the nitroxide is near the methyl terminal or near the ester bond of the 2-acyl chain. The shape is characteristic for spectra of "strongly immobilized probes." Maximum splittings for the lecithin labeled at carbon 16
Biochemistry:
FIG. 2.
Devaux et al.
Spectrum of spin-labeled lecithin I at
Proc. Natl. Acad. Sci. USA 74 (1977)
00.
See legend
to Fig. 1. Vesicles of spin-label I were incubated with exchange protein for 15 min at 300. After cooling to 00, spectra were accumulated for 4 hr. The lower spectrum is the difference between the spectra of
spin-labeled vesicles with and without exchange protein. The distance between two vertical lines represents 10 G.
(spin-label I) is 62 + 1 gausses (G) (1 X 10-4 T). The lecithin labeled at carbon 5 of the 2-acyl chain (spin-label II) gives rise to a slightly bigger splitting, 65 ± 1 G. It is not clear whether this difference indicates an enhanced immobilization or a somewhat more polar environment for spin-label II. Some features, particularly at the low-field part of both difference spectra, could suggest the presence of a minor component that tumbles freely in the water (Fig. 1). Free fatty acid resulting from hydrolysis of the spin-labeled lecithins possibly could give rise to these features although a peak at the low-field region should appear at the high-field region. Addition of free-labeled fatty acids to the exchange protein did not give rise to an immobilized spectrum. This indicates that the immobilized spectra of Fig. 1 reflect the binding of spin-labeled lecithin to the exchange protein. The spectra described above resulted from data accumulation at room temperature. When the exchange protein and vesicles were mixed at room temperature and the spectra were accumulated for 2-3 hr at 00, the characteristic immobilized spectrum was obtained (Fig. 2). The differences between spectra recorded at 200 (Fig. 1 left) and 00 (Fig. 2) very likely reflect differences in rotational mobility of the exchange protein. Effect of ascorbate Ascorbate rapidly destroys the paramagnetism of nitroxides accessible to the aqueous phase (16). In this study it was used
1809
to test the accessibility of the nitroxide group when spin-labeled lecithin is present on the protein. Incubation with ascorbate and ESR measurements were performed at 0° to inhibit the transfer of spin-label between the exchange protein and vesicles. Addition of 2 mM sodium ascorbate had no effect on the appearance of the difference spectrum which, after an accumulation of 3 hr, resembled that of Fig. 2. This shows that the 2-acyl chain of the lecithin molecule bound to the protein is well shielded from the medium. For comparison, addition of 2 mM ascorbate to 5 AM spin-labeled fatty acids in water eliminated the spectrum within 5 to 10 min. Upon interaction with the strong binding sites on albumin, fatty acids with spin-label at different positions in the chain give rise to immobilized spectra similar to the ones observed in this study (5). However, in contrast to what has been observed with the exchange protein, ascorbate rapidly decreased the signal of the spin-labeled fatty acid bound to the albumin molecule (P. F. Devaux, unpublished data). The results described above are consistent with the existence of a cavity in the exchange protein which accommodates the acyl chains of the lecithin molecule. Hsia and Piette (17) used haptens linked to the spin-label by means of acyl chains of different lengths to probe the dimensions of the antigenic site on the antibody. A similar approach could be adopted for the exchange protein to probe the depth of the cavity. Conclusion The ESR spectra of spin-labeled lecithin incorporated into the exchange protein indicate the immobilization of the 2-acyl chain at the methyl and carboxyl terminal ends. This suggests that the 2-acyl chain interacts strongly over its whole length with a hydrophobic cavity in the protein. This study, however, does not provide any insight into the orientation of the lecithin molecule in the protein because both the nitroxide on carbon 16 (analog I) and the nitrogen on carbon 5 (analog II) were inaccessible to ascorbate. The possible small difference in polarity detected by the maximal splitting in the ESR spectra for analog I and II (62 ± 1 and 65 ± 1 G, respectively) could reflect local changes in the cavity. It is likely that the protein exposes a specific binding site for the polar head group of lecithin upon interaction with the interface (H. H. Kamp et al., unpublished data). This, however, does not imply that this binding site is exposed to the medium when the protein is free in solution. Moreover, at this stage it is not known whether the lecithin molecule remains bound to this binding site when it is incorporated into the protein. Independently of the present study, Machida and Ohnishi (personal communication) have shown that spin-labeled lecithin incorporated in the exchange protein from bovine liver gives rise to an immobilized spectrum. The authors thank Mr. J. Westerman for his skillful technical assistance in the purification of the exchange protein. This investigation was supported by research grants from the Del6gation a la Recherche Scientifique et Technique and the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1. Rousselet, A., Colbeau, A., Vignais, P. M. & Devaux, P. F. (1976) Biochim. Biophys. Acta 426,372-384. 2. Demel, R. A., Wirtz, K. W. A., Kamp, H. H., Geurts van Kessel, W. S. M. & van Deenen, L. L. M. (1973) Nature New Biol. 246, 102-105.
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Biochemistry: Devaux et al.
3. Wirtz, K. W. A., Kamp, H. H. & van Deenen, L. L. M. (1972) Biochem. Biophys. Acta 274,606-617. 4. Kamp, H. H., Sprengers, E. D., Westerman, J., Wirtz, K. W. A. & van Deenen, L. L. M. (1975) Biochim. Biophys. Acta 398, 415-423. 5. Morrisett, J. D., Pownall, H. J. & Gotto, A. M. (1975) J. Biol. Chem. 250, 2487-2494. 6. Devaux, P. F., Bienvenue, A., Lauquin, G., Brisson, A. D., Vignais, P. M. & Vignais, P. (1975) Biochemistry 14, 1272-1280. 7. Jost, P. C., Capaldi, R. A., Vanderkooi, G. & Griffith, 0. H. (1973) J. Supramol. Struct. 1, 269-280. 8. Jost, P. C., Griffith, 0. H., Capaldi, R. A. & Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141-152. 9. Hemminga, M. A. & Post, J. F. M. (1976) Biochim. Biophys. Acta 436,222-234. 10. Hesketh, T. R., Smith, G. A., Houslay, M. D., McGill, K. A.,
Proc. Nati. Acad. Sci. USA 74 (1977)
11.
12. 13. 14. 15.
Birdsall, N. J. M., Metcalfe, J. C. & Warren, G. B. (1976) Biochemistry 15, 4145-4151. Kamp, H. H. & Wirtz, K. W. A. (1974) in Methods in Enzymology, eds. Fleischer, S. & Packer, L. (Academic Press, New York), Vol. XXXII, pp. 140-146. Hubbell, W. L. & McConnell, H. M. (1971) J. Am. Chem. Soc. 93,314-323. Davidson, F. M. & Long, C. (1958) Biochem. J. 69, 458-466. Rousselet, A., Guthmann, C., Matricon, J., Bienvenue, A. & Devaux, P. F. (1976) Biochim. Biophys. Acta 426,357-371. van den Besselaar, A. M. H. P., Helmkamp, G. M. & Wirtz, K.
W. A. (1975) Biochemistry 14, 1852-1858. 16. Hubbell, W. L. & McConnell, H. M. (1969) Proc. Natl. Acad. Sci. USA 63, 16-22. 17. Hsia, J. C. & Piette, L. H. (1969) Arch. Biochem. Biophys. 129, 296-307.
Vol. 74, No. 5, pp. 1807-1810, May 1977
Biochemistry
Lipid-protein interaction in the phosphatidylcholine exchange protein (spin-labeled lecithin/electron spin resonance spectroscopy)
PHILIPPE F. DEVAUX*, PETER MOONENt, ALAIN BIENVENUE*, AND KAREL W. A. WIRTZt *
I.B.P.C., 13 rue Pierre et Marie Curie, 75005 Paris, France; and t Laboratory of Biochemistry, State University of Utrecht, Transitorium 3, Padualaan,
Utrecht, The Netherlands
Communicated by Harden M. McConnell, January 10, 1977
MATERIALS AND METHODS Materials. The phosphatidylcholine exchange protein was purified from bovine liver according to established procedures (11). It was stored at -10° in 50% (vol/vol) glycerol at a concentration of approximately 100 gg (4.5 nmol) of protein per ml. Before use, the protein was dialyzed overnight against 20% (wt/vol) sucrose/20 mM sodium acetate/10 mM Tris-HCl, pH 6.6. Protein content was estimated by measuring absorbance at 280 nm (A280 = 0.100 is equivalent to 2.7 nmol of protein per ml). Spin-labeled analogs of lecithin (I and II) were synthesized according to Hubbell and McConnell (12). Their general formula can be written as:
ABSTRACT Incorporation of 2-acyl spin-labeled lecithin into the phosphatidylcholine exchange protein from bovine liver results in an immobilization of the spin-label at the methyl and the carboxyl terminal end of the acyl chain. The nitroxide group on the protein-bound lecithin molecule is not accessible to ascorbate. This suggests that lecithin is buried in a pocket on the protein, which effectively shields the acyl chains from the medium.
The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of spin-labeled lecithin from single bilayer vesicles to rat liver mitochondria (1). Because the protein acts as a carrier, a spin-labeled lecithin is bound to the protein upon removal from the membrane (2). Only those spin-labeled lec-
0 o
CH3-(CH2)mC-(CH2), 0
1
1
CH2-0-C-(CH2)14 -CH3
C O CH
N-0
0
1
CH2-(--P-O-(CH2)2-N(CH3)3 I: m,n = 1,14 LI: mn = 10,3
Phosphatidic acid was prepared from egg yolk lecithin by degradation with phospholipase D according to Davidson and Long (13) and stored as the sodium salt in chloroform at -20°. Vesicles containing the spin-labeled analogs and phosphatidic acid were prepared in sucrose/acetate/Tris buffer as previously described (14). Preparation of Samples. Exchange protein (final concentration, 2-3 AM) was mixed with spin-labeled lecithin vesicles (final concentration, 50-100 ,uM in lipids) containing 2 mol % phosphatidic acid. ESR measurements were performed directly on this mixture and on control samples without exchange pro-
ithin molecules that contained the spin-label on the acyl chain were transferred. Lecithin with the nitroxide in the polar head group was not transferred. This agrees with the notion that the protein contains a binding site that interacts specifically with the lecithin head group exposed at the membrane interface (ref. 3; H. H. Kamp et al., unpublished data). It has previously been demonstrated that the protein binds one molecule of lecithin that cannot be hydrolyzed by phospholipases (4). This indicated that the lecithin molecule was well embedded in the protein. The latter aspect has been elaborated in the present study in which the interaction between the spin-labeled 2-acyl chain of lecithin and the protein has been studied with electron spin resonance (ESR) spectroscopy. It will be shown that measurements of the mobility and accessibility of the nitroxide provide more direct information on the localization of the lecithin molecule in the exchange protein. Similar studies have been performed on the interaction of spin-labeled fatty acids with bovine serum albumin (5). Lipid-protein interactions in membranes have also been studied with spinlabeled lipids (6-10). In general, the latter studies have been complicated by the partitioning of spin-labels between protein and bilayers as well as by nonspecific binding. These problems do not arise when spin-labeled lecithin is bound to the soluble exchange protein.
tein. In some experiments, ESR measurements were performed on the spin-labeled lecithin bound to the protein in the absence of spin-labeled lecithin vesicles. After incubation in sucrose/ acetate/Tris buffer, protein and spin-labeled lecithin vesicles were separated by DEAE-cellulose chromatography. The vesicles that contained 7 mol % phosphatidic acid were retained by the DEAE-cellulose whereas the protein was eluted in the void volume. A chemical reduction of the protein-bound spin-labeled lecithin was attempted at 00 in the presence of 2 mM sodium
ascorbate. Electron Spin Resonance Spectra. ESR measurements were performed on a Varian E 109 spectrometer in 50- or 200-Iu quartz cells. The 200-,ul cell was used for measurements at room
Abbreviation: ESR, electron spin resonance.
1807
1808
Biochemistry: Devaux et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
I
F
0
FIG. 1. Spectra of spin-labeled lecithin I and II at room temperature. The distance between two vertical lines repesents 10 gausses (1 X 10-4 T). Left. The top spectrum corresponds to the accumulated spectra obtained with vesicles of spin label I (nitroxide near the methyl end) in the presence of exchange protein; the middle spectrum is the control in the absence of protein. The mixture contained 500 MM lecithin and 1-2 AM protein; the accumulation period was 3 hr. The lower spectrum is the difference (approximately 10-fold magnification). Right. Same as Left, except for the use of spin label II (nitroxide near the carboxyl end).
temperature and the 50-Al cell for measurements at controlled temperatures. The ESR spectrometer was connected to an In-
tertechnique Didac 4000 multichannel analyzer for data accumulation. Two- to 4-min scans were used, and the accumulation periods were 1-4 hr. These procedures were necessary because of the low protein concentration. The spectra of the spin-labeled lecithin vesicles recorded in the presence and absence of exchange protein were electronically subtracted to obtain the spectrum of the spin-labeled lecithin bound to the exchange protein. RESULTS AND DISCUSSION ESR spectra of spin-labeled lecithin bound to exchange protein The ESR spectrum of vesicles prepared from pure spin-labeled lecithin I or II is a single broad line. This reflects the spin-spin interactions of the lecithin molecules in the bilayer. Upon addition of exchange protein at room temperature, additional structures could be seen superimposed on this broad line. Because the protein concentration is in the order of 2-3 MM, the signal-to-noise ratio of a direct recording is poor. The spectra obtained after data accumulation at room temperature with pure spin-labeled vesicles I and II are shown in Fig. 1. From the direct recording it was apparent that, at room temperature or above, the spectrum characteristic for the exchange proteinvesicle mixture remained constant after 5 min of incubation. On the other hand, when proteins and vesicles were mixed and kept at 00, the superimposed structures became visible only after 1-2 hr. Mixing of protein and vesicles at room temperature followed by recording of the spectra at 00 produced the superimposed lines immediately. These experiments indicate that
the equilibrium governing the interaction of the protein with the spin-labeled vesicles is reached rapidly at room temperature and slowly at 00. The dissociation constant of exchange protein interacting with egg lecithin vesicles that contain 2 mol % phosphatidic acid is 6 X 10-3 M (15). Because the rate of transfer of spin-labeled lecithin I and II was of the same magnitude as that of egg lecithin (1), we assume that the dissociation constant of the exchange protein-spin labeled vesicle complex is of the same order. Given the high value of this constant it can be calculated that virtually no exchange protein is bound to the vesicles. This implies that the difference spectra presented in Fig. 1 represent the unbound protein containing spin-labeled lecithin. In order to obtain direct evidence for this conclusion, exchange protein was mixed with spin-labeled vesicles at room temperature and subsequently separated from these vesicles by DEAE-cellulose chromatography. ESR spectra of the protein present in the eluent were similar to the difference spectra presented in Fig. 1. Because of a more than 50% loss of protein as a result of chromatography, the signal-to-noise ratio of the accumulated spectra was low. A base-line subtraction was necessary. Therefore, in addition to being simpler, the method based on difference spectra gave better results. On the basis of previous studies (ref. 4; H. H. Kamp et al., unpublished data) the unbound protein molecule probably contains one molecule of spin-labeled lecithin after mixing with the spin-labeled vesicles. It is seen from Fig. 1 that the spectrum of the protein-bound spin-labeled lecithin has a similar overall shape independent of whether the nitroxide is near the methyl terminal or near the ester bond of the 2-acyl chain. The shape is characteristic for spectra of "strongly immobilized probes." Maximum splittings for the lecithin labeled at carbon 16
Biochemistry:
FIG. 2.
Devaux et al.
Spectrum of spin-labeled lecithin I at
Proc. Natl. Acad. Sci. USA 74 (1977)
00.
See legend
to Fig. 1. Vesicles of spin-label I were incubated with exchange protein for 15 min at 300. After cooling to 00, spectra were accumulated for 4 hr. The lower spectrum is the difference between the spectra of
spin-labeled vesicles with and without exchange protein. The distance between two vertical lines represents 10 G.
(spin-label I) is 62 + 1 gausses (G) (1 X 10-4 T). The lecithin labeled at carbon 5 of the 2-acyl chain (spin-label II) gives rise to a slightly bigger splitting, 65 ± 1 G. It is not clear whether this difference indicates an enhanced immobilization or a somewhat more polar environment for spin-label II. Some features, particularly at the low-field part of both difference spectra, could suggest the presence of a minor component that tumbles freely in the water (Fig. 1). Free fatty acid resulting from hydrolysis of the spin-labeled lecithins possibly could give rise to these features although a peak at the low-field region should appear at the high-field region. Addition of free-labeled fatty acids to the exchange protein did not give rise to an immobilized spectrum. This indicates that the immobilized spectra of Fig. 1 reflect the binding of spin-labeled lecithin to the exchange protein. The spectra described above resulted from data accumulation at room temperature. When the exchange protein and vesicles were mixed at room temperature and the spectra were accumulated for 2-3 hr at 00, the characteristic immobilized spectrum was obtained (Fig. 2). The differences between spectra recorded at 200 (Fig. 1 left) and 00 (Fig. 2) very likely reflect differences in rotational mobility of the exchange protein. Effect of ascorbate Ascorbate rapidly destroys the paramagnetism of nitroxides accessible to the aqueous phase (16). In this study it was used
1809
to test the accessibility of the nitroxide group when spin-labeled lecithin is present on the protein. Incubation with ascorbate and ESR measurements were performed at 0° to inhibit the transfer of spin-label between the exchange protein and vesicles. Addition of 2 mM sodium ascorbate had no effect on the appearance of the difference spectrum which, after an accumulation of 3 hr, resembled that of Fig. 2. This shows that the 2-acyl chain of the lecithin molecule bound to the protein is well shielded from the medium. For comparison, addition of 2 mM ascorbate to 5 AM spin-labeled fatty acids in water eliminated the spectrum within 5 to 10 min. Upon interaction with the strong binding sites on albumin, fatty acids with spin-label at different positions in the chain give rise to immobilized spectra similar to the ones observed in this study (5). However, in contrast to what has been observed with the exchange protein, ascorbate rapidly decreased the signal of the spin-labeled fatty acid bound to the albumin molecule (P. F. Devaux, unpublished data). The results described above are consistent with the existence of a cavity in the exchange protein which accommodates the acyl chains of the lecithin molecule. Hsia and Piette (17) used haptens linked to the spin-label by means of acyl chains of different lengths to probe the dimensions of the antigenic site on the antibody. A similar approach could be adopted for the exchange protein to probe the depth of the cavity. Conclusion The ESR spectra of spin-labeled lecithin incorporated into the exchange protein indicate the immobilization of the 2-acyl chain at the methyl and carboxyl terminal ends. This suggests that the 2-acyl chain interacts strongly over its whole length with a hydrophobic cavity in the protein. This study, however, does not provide any insight into the orientation of the lecithin molecule in the protein because both the nitroxide on carbon 16 (analog I) and the nitrogen on carbon 5 (analog II) were inaccessible to ascorbate. The possible small difference in polarity detected by the maximal splitting in the ESR spectra for analog I and II (62 ± 1 and 65 ± 1 G, respectively) could reflect local changes in the cavity. It is likely that the protein exposes a specific binding site for the polar head group of lecithin upon interaction with the interface (H. H. Kamp et al., unpublished data). This, however, does not imply that this binding site is exposed to the medium when the protein is free in solution. Moreover, at this stage it is not known whether the lecithin molecule remains bound to this binding site when it is incorporated into the protein. Independently of the present study, Machida and Ohnishi (personal communication) have shown that spin-labeled lecithin incorporated in the exchange protein from bovine liver gives rise to an immobilized spectrum. The authors thank Mr. J. Westerman for his skillful technical assistance in the purification of the exchange protein. This investigation was supported by research grants from the Del6gation a la Recherche Scientifique et Technique and the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1. Rousselet, A., Colbeau, A., Vignais, P. M. & Devaux, P. F. (1976) Biochim. Biophys. Acta 426,372-384. 2. Demel, R. A., Wirtz, K. W. A., Kamp, H. H., Geurts van Kessel, W. S. M. & van Deenen, L. L. M. (1973) Nature New Biol. 246, 102-105.
1810
Biochemistry: Devaux et al.
3. Wirtz, K. W. A., Kamp, H. H. & van Deenen, L. L. M. (1972) Biochem. Biophys. Acta 274,606-617. 4. Kamp, H. H., Sprengers, E. D., Westerman, J., Wirtz, K. W. A. & van Deenen, L. L. M. (1975) Biochim. Biophys. Acta 398, 415-423. 5. Morrisett, J. D., Pownall, H. J. & Gotto, A. M. (1975) J. Biol. Chem. 250, 2487-2494. 6. Devaux, P. F., Bienvenue, A., Lauquin, G., Brisson, A. D., Vignais, P. M. & Vignais, P. (1975) Biochemistry 14, 1272-1280. 7. Jost, P. C., Capaldi, R. A., Vanderkooi, G. & Griffith, 0. H. (1973) J. Supramol. Struct. 1, 269-280. 8. Jost, P. C., Griffith, 0. H., Capaldi, R. A. & Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141-152. 9. Hemminga, M. A. & Post, J. F. M. (1976) Biochim. Biophys. Acta 436,222-234. 10. Hesketh, T. R., Smith, G. A., Houslay, M. D., McGill, K. A.,
Proc. Nati. Acad. Sci. USA 74 (1977)
11.
12. 13. 14. 15.
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