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A convenient and sensitive fluorescence assay for phospholipid vesicles using diphenylhexatriene
ANALYTICAL
BIOCHEMISTRY
A Convenient
88,
203-211 (1978)
and Sensitive Fluorescence Phospholipid Vesicles Using Diphenylhexatriene E. LONDON
Section
of Biochemistry,
Assay for
AND G. W. FEIGENSON
Moleculur and Cell Biology, Clark Ithaca, New York 14853
Hall,
Cornell
University,
Received July 25, 1977; accepted February 27, 1978 When phospholipid vesicles are added to an aqueous solution of 1,6-diphenyl1,3,5-hexatriene (DPH) a fluorescence enhancement of up to several hundredfold is observed which can be used for a determination of phospholipid concentration. Fluorescence enhancement of 2 pM DPH is proportional to the phospholipid concentration over a wide range. As little as 0.7 nmol (-0.5 pg of phospholipid) can be determined to within + 10%. The fluorescence is a function of the type of phospholipid used, salt concentration, and time of incubation. Protein and detergents also enhance DPH fluorescence but to a much smaller extent. Optimal conditions for the assay are presented. Use of this assay to detect phospholipid vesicles fractionated by size on a Sepharose 4B column is illustrated. In this case the method compares favorably to more classical methods of analysis in terms of sensitivity, accuracy, and time involved.
Many fluorescent molecules exhibit an increase in fluorescence upon binding to protein or upon entering phospholipid membranes. ANS (l-5),’ NPN (6,7), and DPH (8,9) are well-known examples. DPH has been used in studies of membrane motion which utilize the polarization of its fluorescence [see, for example, Ref. (8- 1 l)]. The membrane probes ANS (12,13) and NPN (14) have been used to assay for phospholipid on thinlayer chromatograms. The factors influencing the fluorescence of ANS are complex (4,5), at least in part because of its charge and its high solubility in water. The fluorescence of NPN in water is appreciable, and the fluorescence enhancement upon binding to liposomes is only 20-fold (7). DPH is more suitable for fluorescence assay of phospholipid vesicles because of its low background fluorescence in water and because it is uncharged and, so, should be relatively unaffected by the presence of ions and other molecules dissolved in water. ’ Abbreviations used: DPH, 1,6-diphenyl-1,3,Shexatriene; ANS, I-anihno-l-naphthalenesulfonic acid; NPN, N-phenyl-1-naphthylamine; Pi. inorganic orthophosphate; PC, phosphatidylcholine; BSA, bovine serum albumin; tic, thin-layer chromatography; cmc, critical micelle concentration. 203
0003-2697/78/0881-0203$02.00/O Copyright 0 1978 by Academic Press, Inc. All tights of reproduction in any form reserved.
204
LONDONANDFEIGENSON MATERIALS
AND METHODS
Dimyristoyl PC, BSA grade “essentially fatty acid free,” Triton X-100, and Sepharose 4B were purchased from Sigma. DPH was purchased from Aldrich and used without further purification. Crude soy phospholipids were purchased from Associated Concentrates. Egg PC was isolated by the method of Small et al. (15). The dimyristoyl and egg PC were of >98% purity by tic. Small single bilayer vesicles were formed by sonication for about 30 min at 40°C using a bath sonicator (Laboratory Supplies, Co., Inc.). Banghasomes (coarse multilamellar liposomes) were produced by adding water to a dried film of phospholipid in a test tube and then shaking until a uniform dispersion was produced. Surface tension was measured with a Du Nouy tensiometer. Pi was analyzed by the method of Bartlett (16) after the digestion of phospholipid according to Morrison (17) with some modification. Optical density was measured with a Beckman 25 spectrophotometer. Fluorescence measurements were made with a Perkin-Elmer MPF-3 spectrofluorimeter operating in ratio mode. The excitation wavelength was 365 nm, and emission wavelength was 460 nm. The excitation and emission slits were set at nominal bandwidths of 5 and 10 nm, respectively. The fluorescence scale in the figures is arbitrary, and fluorescence values are comparable only within each graph. The usual protocol for assay was as follows: 2 ~1 of 3 mM DPH in tetrahydrofuran was added to a solution of 3 ml of water and 1 to 50 ~1 of phospholipid vesicles. Tubes were incubated in the dark for 40-60 min at 40°C and then fluorescence was measured while exposing the sample to the excitation beam for 5 sec. Each point in a figure is the result of a single determination. Exposure of the sample to room light for over a minute was avoided once DPH had been added. A fresh solution of 3 mM DPH was prepared monthly and stored in the dark. More detail may be found under Results and in figure legends. RESULTS
The dependence of DPH fluorescence on phospholipid concentration is shown in Figs. 1A and B. With the protocol reported under Materials and Methods less than 5 nmol can be detected. As shown in Fig. 1B the fluorescence is linear up to 100 nmol. In this range errors caused by light scattering are negligible. Using a modified protocol, with only a 0.3-ml total volume in a semimicrocuvette sensitivity can be improved IO-fold (Fig. 1A). In Fig. IA the standard error of estimate is only 0.07 nmol, thus 0.7 nmol (-0.5 pg of phospholipid) can be determined to within f 10%. The dependence of the linear range on DPH concentration is shown in Fig. 2. With up to 30 nmol of dimyristoyl PC, 0.1 to 4 pM DPH gives identical results. However as can be seen in the inset to Fig. 2, at
ASSAY FOR PHOSPHOLIPID
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FIG. 1. Dependence of fluorescence, F, on phospholipid vesicle concentration. Sonicated dimyristoyl PC was incubated at various concentrations: (A) with 0.3 ml of 2 pM DPH for 45 min at 40°C and (B) with 3 ml of 2 pM DPH for 45 min at 40°C. Other details are given under Materials and Methods.
higher concentrations of phospholipid fluorescence is no longer independent of DPH. The lower the concentration of DPH, the lower is the upper limit of linearity. The species and physical form of the phospholipid used have an effect on the fluorescence as shown in Fig. 3A. Use of banghasomes of dimyristoyl PC, sonicated egg PC, or sonicated mixed phospholipids results in 20 to 40% less fluorescence than is observed with sonicated dimyristoyl PC. The presence of ions also affects fluorescence. CaCl, at 10 mM or NaCl at 100 mM depresses the fluorescence by -20% without affecting linearity (Fig. 3B). The length of the period of incubation of DPH with the vesicles is another important variable. As seen in Figs. 4A and B for sonicated dimyristoyl PC vesicles, fluorescence reaches a maximum value after 30 min. For different phospholipids, and in the presence of salts, the time to reach the fluorescence maximum occurs within 20 to 60 min except in the case of egg PC where it occurs after 5 min of incubation, as shown in Figs. 4A and B. The cause of the slow fluorescent decay at longer times is unclear. The effect of protein or detergent on the DPH fluorescence was determined. As shown in Fig. 5, BSA and Triton X-100 enhance DPH fluorescence by only a few percent of the enhancement from phospholipid at similar concentrations. The fluorescence is linear with BSA and with Triton X-100 at low concentrations. At higher concentrations with fluorescence jumps dramatically for Triton X- 100. This should occur at the
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mmoles)
FIG. 2. Dependence of fluorescence on phospholipid concentration tions of DPH. Sonicated dimyristoyl PC vesicles were incubated with 3 ml of (0) 0.1, (x) 0.5, (0) 2, and (A) 4 pM DPH.
PC
at various concentrafor 4.5 min at 40°C
,nmol..,
FIG. 3.(A) Dependence of fluorescence on species or physical form of phospholipid. Samples contained various concentrations of (0) sonicated dimyristoyl PC, (x) unsonicated dimyristoyl PC banghasomes, (0) sonicated egg PC, or (*) sonicated mixed soy phospholipids. Each was incubated with 3 ml of 2 FM DPH for 45 min at 40°C. (B) Effect of salts on fluorescence. Samples contained sonicated dimyristoyl PC together with (0) no added salt, (A) 100 mM NaCI, or (A) 10 mM CaCl,. Each was incubated with 3 ml of 2 PM DPH for 45 min at 40°C.
ASSAY
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FIG. 4. Dependence of fluorescence on length of incubation. Fluorescence was measured at various times after the start of the incubation period (see Materials and Methods). (A) Dependence on type of phospholipid. Samples contained (0) 78.5 nmol of sonicated dimyristoyl PC, (x) 74.5 nmol of unsonicated dimyristoyl PC banghasomes, or (0) 68.0 nmol of sonicated egg PC. Each was incubated with 3 ml of 2 PM DPH at 40°C. (B) Effect of salts. Samples contained (0) no added salt, (A) 100 mM NaCl, or (A) 10 mM CaCl, and 59.5 nmol of sonicated dimyristoyl PC. Each was incubated with 3 ml of 2 pM DPH at 40°C.
cmc of Triton X-100 where micelle formation provides a hydrophobic environment for the DPH. In fact, we found that the cmc determined by surface tension vs Triton X-100 concentration agreed with the value from fluorescence. One attractive possibility is to use this assay for the determination of
FIG. 5. Enhancement of DPH fluorescence resulting from protein or detergent. Samples contained various concentrations of (0) sonicated dimyristoyl PC vesicles, (0) BSA. or (x) Triton X-100. Each was incubated with 3 ml of 2 pM DPH for 45 min at 40°C.
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phospholipid vesicles or membranes fractionated by column chromatography. An example of this use is presented in Fig. 6. The phospholipid vesicles were fractionated on a 40 x 1J-cm column of Sepharose 4B. The void volume was determined with a sample of dimyristoyl PC banghasomes. One-half milliliter of - 16 pmol/ml of sonicated dimyristoyl PC vesicles was applied to the column. Fractions of 0.85 ml were collected. The optical density prohle at 260 nm indicates that about 20% of all phospholipid appears in the void volume and the maximum concentration in the main peak of phospholipid appears in fraction 50, which is not in agreement with either Pi analysis or DPH fluorescence assay. In contrast to the optical density, the Pi analysis and the DPH fluorescence assay are in good agreement as to the magnitude of the void volume peak and the location and shape of the main peak. d
A.
FRACTION
NUMBER
FIG. 6. Fractionation of 7.9 pmol of sonicated dimyristoyl PC vesicles on a 40 x 1.5 cm column of Sepharose 4B. The fractions were monitored by (A) optical density at 260 nm, (B) P, analysis, or (C) DPH fluorescence enhancement by phospholipid. For P, analysis 200 ~1 of each fraction was used. For fluorescence 50 ~1 of each fraction was added to 3 ml of 2 /.bM DPH and then incubated for 60 min at 40°C.
ASSAY FOR PHOSPHOLIPID
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DISCUSSION
This assay for phospholipid vesicles is rapid and sensitive, requiring -30 min for sample incubation and detecting -0.7 nmol with 10% error. A maximum sensitivity is achieved by using a fluorescence semimicrocuvette, which requires only 0.3 ml of sample. Previous workers have noted that the increase in DPH fluorescence upon addition of phospholipid to an aqueous DPH solution is caused by the entry of the DPH molecule into the bilayer (9,lO). The present observations imply that this process is not governed by a partition coefficient (K) relating DPH concentration to the volumes of the lipid and the aqueous phases as follows: DPHii,id/DPH,,
= K(Volumeir,i,JVolume.,).
[II
If DPH did obey such a relationship, then doubling the total DPH concentration would double the value of DPHirpid. This would be true because doubling total DPH would not affect the right-hand side of [ 11, and to maintain the same ratio DPHirpid and DPH,, must each double. This is not observed. Instead it is possible to saturate phospholipid with DPH so that DPH fluorescence remains constant as more DPH is added, as can be observed in Fig. 2 for PC concentrations 5 10 PM. At saturation the DPH/PC ratio is as low as 0.1 PM/IO PM, so that relatively few DPH molecules bind to each vesicle at saturation. The “binding site” or vacancy occupied by the DPH molecule must involve many PC molecules. The limit of linearity of the assay is determined by the DPH/PC ratio. At concentrations of DPH where it is in excess relative to phospholipid (i.e., PC is saturated with DPH) fluorescence will be linear with PC concentration. For this reason high DPH concentrations are advantageous. However, the DPH concentration used was generally 2 ,UM despite the fact that with higher concentrations the upper limit of linearity is extended. Above 2 PM the absorbance of DPH becomes significant, producing an inner filter effect which reduces the expected fluorescence. More important is the fact that the solubility of DPH in water is limited, and turbidity becomes significant when greater amounts of DPH in tetrahydrofuran are injected into water. This turbidity is not observed when tetrahydrofuran alone is injected. In fact, we have observed that even at 2 F’M DPH there was significant turbidity as measured by 90” light scattering at 420 nm, where DPH does not absorb. Thus, at the concentrations studied, DPH is not in true solution, but in some colloidal form. It is appropriate to comment on the various factors which influence the DPH fluorescence enhancement. The temperature of incubation should be at least 10°C above the phase transition temperature of the phospholipid, if it has a phase transition, since DPH penetrates the more mobile
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liquid crystal phase more rapidly (IO), and thus fluorescence reaches its final value more rapidly. Ideally, the samples should be maintained at constant temperature while measuring fluorescence to eliminate the possibility of a significant error from the temperature dependence of quantum yield and of DPH binding to the vesicles. Figure 3A shows that the DPH fluorescence enhancement depends upon the species of phospholipid and the degree of saturation of the fatty acid chains. Therefore a standard curve of fluorescence enhancement should be prepared using the particular phospholipids which are to be assayed. Others (12,14) have observed that ANS and NPN fluorescence enhancements on tic plates also depend on phospholipid species and chain unsaturation. The dependence of the fluorescence enhancement upon the type of phospholipid used may involve the number of DPH molecules which can enter the vesicles of the different types of phospholipids. Another possibility is that quantum yield of DPH is affected by these differences. The fluorescence intensities of DPH in dimyristoyl PC banghasomes and in sonicated vesicles may converge to the same value at very long times, when DPH has completely penetrated the multilayered banghasomes, or else differences in bilayer structure might be responsible for the different fluorescent responses observed. Ions reduce DPH fluorescence enhancement, although the mechanism is uncertain. ANS fluorescence shows a much greater dependence on ion concentration (4). The effect of BSA and Triton X-100 on DPH fluorescence is presumably similar in mechanism to the effect of phospholipid. The differences in fluorescence enhancement between these substances and PC vesicles may reflect differences in the amount of DPH bound per microgram or differences in quantum yield of DPH in a protein, detergent, or lipid environment. It is interesting that this method is a sensitive way to measure the cmc of Triton X-100, as noted under Results. The most promising application of this technique is to assay for phospholipid vesicles fractionated by column chromatography. The similarity of the column profiles by Pi analysis or DPH fluorescence enhancement indicates that the DPH fluorescence enhancement is proportional to the phospholipid concentration and that fluorescence enhancement is not a strong function of vesicle size. The sensitivity of this assay is comparable to that of Pi analysis. It is also nondestructive. Although optical density measurements are also convenient and nondestructive, differential light scattering by vesicles of different size prevents the use of optical density as a quantitative measure of phospholipid concentration as has been noted previously [see, for example, Ref. (18)]. Radioactivity measurement can be the most sensitive technique, but radioactively labeled phospholipid is required. Overall, the convenience, accuracy, and sensitivity of this DPH fluorescence assay make it ideal for application to column chromatography of model or real membranes.
ASSAY FOR PHOSPHOLIPID
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ACKNOWLEDGMENTS The authors wish to thank Dr. Don Zilversmit for the loan of the Du Nouy tensiometer and Ms. Diane Bachman and Mr. David Balitz for assistance. This work was supported by National Institutes of Health Grant HL 18255. E. L. was supported in part by a Biophysics Fellowship from Cornell University.
REFERENCES 1. Fildes, J. E., Laurence, D. J. R., and Rees, V. H. (1954) Biochem. J. 56, xxxi-xxxii. Stryer, L. (1965) J. Mol. Biol. 13, 482-495. Freedman, R. B., and Radda, G. K. (1969) FEBS Lett. 3, 150-152. 4. Vanderkooi, J., and Martinosi, A. (1969) Arch. Biochem. Biophys. 133, 153-163. 5. Radda, G. K., and Vanderkooi, J. (1972) Eiochim. Eiophys. Acta 265, 509-549. 6. Overath, P., and Trauble, H. (1973) Biochemistry 12, 2625-2634. 7. Trluble, H.. and Overath, P. (1973) Biochim. Biophys. Acta 307, 491-512. 8. Shinitzky, M., and Inbar, M. (1974) J. Mol. Biol. 85, 603-615. 9. Shinitzky, M., and Barenholz, Y. (1974) J. Biol. Chem. 249, 2652-2657. 10. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976)Biochemistry l&4521-4528. 11. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976)Biochemistry 15, 4529-4537. 12. Heyneman, R. A., Bernard, D. M., and Vercauteren, R. E. (1972) J. Chromatogr. 2. 3.
68, 285-288.
13. Gitler, C. (1972) Anal. Biochem. 50, 324-325. 14. Bernard, D. M., and Vercauteren, R. E. (1976) J. Chromatogr. 120, 211-212. 15. Small, D. M., Bourges, M. C., and Dervichian, D. G. (1966) Biochim. Biophys. Acfa 125, 563-580.
16. Bartlett, G. R. (1959)5. Biol. Chem. 234, 466-468. 17. Morrison, W. R. (1964) Anal. Biochem. 7, 218-224. 18. Huang, C., and Charlton. J. P. (1972)Biochem. Biophys. Res. Commun. 46, 1660-1666.
BIOCHEMISTRY
A Convenient
88,
203-211 (1978)
and Sensitive Fluorescence Phospholipid Vesicles Using Diphenylhexatriene E. LONDON
Section
of Biochemistry,
Assay for
AND G. W. FEIGENSON
Moleculur and Cell Biology, Clark Ithaca, New York 14853
Hall,
Cornell
University,
Received July 25, 1977; accepted February 27, 1978 When phospholipid vesicles are added to an aqueous solution of 1,6-diphenyl1,3,5-hexatriene (DPH) a fluorescence enhancement of up to several hundredfold is observed which can be used for a determination of phospholipid concentration. Fluorescence enhancement of 2 pM DPH is proportional to the phospholipid concentration over a wide range. As little as 0.7 nmol (-0.5 pg of phospholipid) can be determined to within + 10%. The fluorescence is a function of the type of phospholipid used, salt concentration, and time of incubation. Protein and detergents also enhance DPH fluorescence but to a much smaller extent. Optimal conditions for the assay are presented. Use of this assay to detect phospholipid vesicles fractionated by size on a Sepharose 4B column is illustrated. In this case the method compares favorably to more classical methods of analysis in terms of sensitivity, accuracy, and time involved.
Many fluorescent molecules exhibit an increase in fluorescence upon binding to protein or upon entering phospholipid membranes. ANS (l-5),’ NPN (6,7), and DPH (8,9) are well-known examples. DPH has been used in studies of membrane motion which utilize the polarization of its fluorescence [see, for example, Ref. (8- 1 l)]. The membrane probes ANS (12,13) and NPN (14) have been used to assay for phospholipid on thinlayer chromatograms. The factors influencing the fluorescence of ANS are complex (4,5), at least in part because of its charge and its high solubility in water. The fluorescence of NPN in water is appreciable, and the fluorescence enhancement upon binding to liposomes is only 20-fold (7). DPH is more suitable for fluorescence assay of phospholipid vesicles because of its low background fluorescence in water and because it is uncharged and, so, should be relatively unaffected by the presence of ions and other molecules dissolved in water. ’ Abbreviations used: DPH, 1,6-diphenyl-1,3,Shexatriene; ANS, I-anihno-l-naphthalenesulfonic acid; NPN, N-phenyl-1-naphthylamine; Pi. inorganic orthophosphate; PC, phosphatidylcholine; BSA, bovine serum albumin; tic, thin-layer chromatography; cmc, critical micelle concentration. 203
0003-2697/78/0881-0203$02.00/O Copyright 0 1978 by Academic Press, Inc. All tights of reproduction in any form reserved.
204
LONDONANDFEIGENSON MATERIALS
AND METHODS
Dimyristoyl PC, BSA grade “essentially fatty acid free,” Triton X-100, and Sepharose 4B were purchased from Sigma. DPH was purchased from Aldrich and used without further purification. Crude soy phospholipids were purchased from Associated Concentrates. Egg PC was isolated by the method of Small et al. (15). The dimyristoyl and egg PC were of >98% purity by tic. Small single bilayer vesicles were formed by sonication for about 30 min at 40°C using a bath sonicator (Laboratory Supplies, Co., Inc.). Banghasomes (coarse multilamellar liposomes) were produced by adding water to a dried film of phospholipid in a test tube and then shaking until a uniform dispersion was produced. Surface tension was measured with a Du Nouy tensiometer. Pi was analyzed by the method of Bartlett (16) after the digestion of phospholipid according to Morrison (17) with some modification. Optical density was measured with a Beckman 25 spectrophotometer. Fluorescence measurements were made with a Perkin-Elmer MPF-3 spectrofluorimeter operating in ratio mode. The excitation wavelength was 365 nm, and emission wavelength was 460 nm. The excitation and emission slits were set at nominal bandwidths of 5 and 10 nm, respectively. The fluorescence scale in the figures is arbitrary, and fluorescence values are comparable only within each graph. The usual protocol for assay was as follows: 2 ~1 of 3 mM DPH in tetrahydrofuran was added to a solution of 3 ml of water and 1 to 50 ~1 of phospholipid vesicles. Tubes were incubated in the dark for 40-60 min at 40°C and then fluorescence was measured while exposing the sample to the excitation beam for 5 sec. Each point in a figure is the result of a single determination. Exposure of the sample to room light for over a minute was avoided once DPH had been added. A fresh solution of 3 mM DPH was prepared monthly and stored in the dark. More detail may be found under Results and in figure legends. RESULTS
The dependence of DPH fluorescence on phospholipid concentration is shown in Figs. 1A and B. With the protocol reported under Materials and Methods less than 5 nmol can be detected. As shown in Fig. 1B the fluorescence is linear up to 100 nmol. In this range errors caused by light scattering are negligible. Using a modified protocol, with only a 0.3-ml total volume in a semimicrocuvette sensitivity can be improved IO-fold (Fig. 1A). In Fig. IA the standard error of estimate is only 0.07 nmol, thus 0.7 nmol (-0.5 pg of phospholipid) can be determined to within f 10%. The dependence of the linear range on DPH concentration is shown in Fig. 2. With up to 30 nmol of dimyristoyl PC, 0.1 to 4 pM DPH gives identical results. However as can be seen in the inset to Fig. 2, at
ASSAY FOR PHOSPHOLIPID
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FIG. 1. Dependence of fluorescence, F, on phospholipid vesicle concentration. Sonicated dimyristoyl PC was incubated at various concentrations: (A) with 0.3 ml of 2 pM DPH for 45 min at 40°C and (B) with 3 ml of 2 pM DPH for 45 min at 40°C. Other details are given under Materials and Methods.
higher concentrations of phospholipid fluorescence is no longer independent of DPH. The lower the concentration of DPH, the lower is the upper limit of linearity. The species and physical form of the phospholipid used have an effect on the fluorescence as shown in Fig. 3A. Use of banghasomes of dimyristoyl PC, sonicated egg PC, or sonicated mixed phospholipids results in 20 to 40% less fluorescence than is observed with sonicated dimyristoyl PC. The presence of ions also affects fluorescence. CaCl, at 10 mM or NaCl at 100 mM depresses the fluorescence by -20% without affecting linearity (Fig. 3B). The length of the period of incubation of DPH with the vesicles is another important variable. As seen in Figs. 4A and B for sonicated dimyristoyl PC vesicles, fluorescence reaches a maximum value after 30 min. For different phospholipids, and in the presence of salts, the time to reach the fluorescence maximum occurs within 20 to 60 min except in the case of egg PC where it occurs after 5 min of incubation, as shown in Figs. 4A and B. The cause of the slow fluorescent decay at longer times is unclear. The effect of protein or detergent on the DPH fluorescence was determined. As shown in Fig. 5, BSA and Triton X-100 enhance DPH fluorescence by only a few percent of the enhancement from phospholipid at similar concentrations. The fluorescence is linear with BSA and with Triton X-100 at low concentrations. At higher concentrations with fluorescence jumps dramatically for Triton X- 100. This should occur at the
206
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FEIGENSON
mmoles)
FIG. 2. Dependence of fluorescence on phospholipid concentration tions of DPH. Sonicated dimyristoyl PC vesicles were incubated with 3 ml of (0) 0.1, (x) 0.5, (0) 2, and (A) 4 pM DPH.
PC
at various concentrafor 4.5 min at 40°C
,nmol..,
FIG. 3.(A) Dependence of fluorescence on species or physical form of phospholipid. Samples contained various concentrations of (0) sonicated dimyristoyl PC, (x) unsonicated dimyristoyl PC banghasomes, (0) sonicated egg PC, or (*) sonicated mixed soy phospholipids. Each was incubated with 3 ml of 2 FM DPH for 45 min at 40°C. (B) Effect of salts on fluorescence. Samples contained sonicated dimyristoyl PC together with (0) no added salt, (A) 100 mM NaCI, or (A) 10 mM CaCl,. Each was incubated with 3 ml of 2 PM DPH for 45 min at 40°C.
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FIG. 4. Dependence of fluorescence on length of incubation. Fluorescence was measured at various times after the start of the incubation period (see Materials and Methods). (A) Dependence on type of phospholipid. Samples contained (0) 78.5 nmol of sonicated dimyristoyl PC, (x) 74.5 nmol of unsonicated dimyristoyl PC banghasomes, or (0) 68.0 nmol of sonicated egg PC. Each was incubated with 3 ml of 2 PM DPH at 40°C. (B) Effect of salts. Samples contained (0) no added salt, (A) 100 mM NaCl, or (A) 10 mM CaCl, and 59.5 nmol of sonicated dimyristoyl PC. Each was incubated with 3 ml of 2 pM DPH at 40°C.
cmc of Triton X-100 where micelle formation provides a hydrophobic environment for the DPH. In fact, we found that the cmc determined by surface tension vs Triton X-100 concentration agreed with the value from fluorescence. One attractive possibility is to use this assay for the determination of
FIG. 5. Enhancement of DPH fluorescence resulting from protein or detergent. Samples contained various concentrations of (0) sonicated dimyristoyl PC vesicles, (0) BSA. or (x) Triton X-100. Each was incubated with 3 ml of 2 pM DPH for 45 min at 40°C.
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phospholipid vesicles or membranes fractionated by column chromatography. An example of this use is presented in Fig. 6. The phospholipid vesicles were fractionated on a 40 x 1J-cm column of Sepharose 4B. The void volume was determined with a sample of dimyristoyl PC banghasomes. One-half milliliter of - 16 pmol/ml of sonicated dimyristoyl PC vesicles was applied to the column. Fractions of 0.85 ml were collected. The optical density prohle at 260 nm indicates that about 20% of all phospholipid appears in the void volume and the maximum concentration in the main peak of phospholipid appears in fraction 50, which is not in agreement with either Pi analysis or DPH fluorescence assay. In contrast to the optical density, the Pi analysis and the DPH fluorescence assay are in good agreement as to the magnitude of the void volume peak and the location and shape of the main peak. d
A.
FRACTION
NUMBER
FIG. 6. Fractionation of 7.9 pmol of sonicated dimyristoyl PC vesicles on a 40 x 1.5 cm column of Sepharose 4B. The fractions were monitored by (A) optical density at 260 nm, (B) P, analysis, or (C) DPH fluorescence enhancement by phospholipid. For P, analysis 200 ~1 of each fraction was used. For fluorescence 50 ~1 of each fraction was added to 3 ml of 2 /.bM DPH and then incubated for 60 min at 40°C.
ASSAY FOR PHOSPHOLIPID
VESICLES
209
DISCUSSION
This assay for phospholipid vesicles is rapid and sensitive, requiring -30 min for sample incubation and detecting -0.7 nmol with 10% error. A maximum sensitivity is achieved by using a fluorescence semimicrocuvette, which requires only 0.3 ml of sample. Previous workers have noted that the increase in DPH fluorescence upon addition of phospholipid to an aqueous DPH solution is caused by the entry of the DPH molecule into the bilayer (9,lO). The present observations imply that this process is not governed by a partition coefficient (K) relating DPH concentration to the volumes of the lipid and the aqueous phases as follows: DPHii,id/DPH,,
= K(Volumeir,i,JVolume.,).
[II
If DPH did obey such a relationship, then doubling the total DPH concentration would double the value of DPHirpid. This would be true because doubling total DPH would not affect the right-hand side of [ 11, and to maintain the same ratio DPHirpid and DPH,, must each double. This is not observed. Instead it is possible to saturate phospholipid with DPH so that DPH fluorescence remains constant as more DPH is added, as can be observed in Fig. 2 for PC concentrations 5 10 PM. At saturation the DPH/PC ratio is as low as 0.1 PM/IO PM, so that relatively few DPH molecules bind to each vesicle at saturation. The “binding site” or vacancy occupied by the DPH molecule must involve many PC molecules. The limit of linearity of the assay is determined by the DPH/PC ratio. At concentrations of DPH where it is in excess relative to phospholipid (i.e., PC is saturated with DPH) fluorescence will be linear with PC concentration. For this reason high DPH concentrations are advantageous. However, the DPH concentration used was generally 2 ,UM despite the fact that with higher concentrations the upper limit of linearity is extended. Above 2 PM the absorbance of DPH becomes significant, producing an inner filter effect which reduces the expected fluorescence. More important is the fact that the solubility of DPH in water is limited, and turbidity becomes significant when greater amounts of DPH in tetrahydrofuran are injected into water. This turbidity is not observed when tetrahydrofuran alone is injected. In fact, we have observed that even at 2 F’M DPH there was significant turbidity as measured by 90” light scattering at 420 nm, where DPH does not absorb. Thus, at the concentrations studied, DPH is not in true solution, but in some colloidal form. It is appropriate to comment on the various factors which influence the DPH fluorescence enhancement. The temperature of incubation should be at least 10°C above the phase transition temperature of the phospholipid, if it has a phase transition, since DPH penetrates the more mobile
210
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AND
FEIGENSON
liquid crystal phase more rapidly (IO), and thus fluorescence reaches its final value more rapidly. Ideally, the samples should be maintained at constant temperature while measuring fluorescence to eliminate the possibility of a significant error from the temperature dependence of quantum yield and of DPH binding to the vesicles. Figure 3A shows that the DPH fluorescence enhancement depends upon the species of phospholipid and the degree of saturation of the fatty acid chains. Therefore a standard curve of fluorescence enhancement should be prepared using the particular phospholipids which are to be assayed. Others (12,14) have observed that ANS and NPN fluorescence enhancements on tic plates also depend on phospholipid species and chain unsaturation. The dependence of the fluorescence enhancement upon the type of phospholipid used may involve the number of DPH molecules which can enter the vesicles of the different types of phospholipids. Another possibility is that quantum yield of DPH is affected by these differences. The fluorescence intensities of DPH in dimyristoyl PC banghasomes and in sonicated vesicles may converge to the same value at very long times, when DPH has completely penetrated the multilayered banghasomes, or else differences in bilayer structure might be responsible for the different fluorescent responses observed. Ions reduce DPH fluorescence enhancement, although the mechanism is uncertain. ANS fluorescence shows a much greater dependence on ion concentration (4). The effect of BSA and Triton X-100 on DPH fluorescence is presumably similar in mechanism to the effect of phospholipid. The differences in fluorescence enhancement between these substances and PC vesicles may reflect differences in the amount of DPH bound per microgram or differences in quantum yield of DPH in a protein, detergent, or lipid environment. It is interesting that this method is a sensitive way to measure the cmc of Triton X-100, as noted under Results. The most promising application of this technique is to assay for phospholipid vesicles fractionated by column chromatography. The similarity of the column profiles by Pi analysis or DPH fluorescence enhancement indicates that the DPH fluorescence enhancement is proportional to the phospholipid concentration and that fluorescence enhancement is not a strong function of vesicle size. The sensitivity of this assay is comparable to that of Pi analysis. It is also nondestructive. Although optical density measurements are also convenient and nondestructive, differential light scattering by vesicles of different size prevents the use of optical density as a quantitative measure of phospholipid concentration as has been noted previously [see, for example, Ref. (18)]. Radioactivity measurement can be the most sensitive technique, but radioactively labeled phospholipid is required. Overall, the convenience, accuracy, and sensitivity of this DPH fluorescence assay make it ideal for application to column chromatography of model or real membranes.
ASSAY FOR PHOSPHOLIPID
VESICLES
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ACKNOWLEDGMENTS The authors wish to thank Dr. Don Zilversmit for the loan of the Du Nouy tensiometer and Ms. Diane Bachman and Mr. David Balitz for assistance. This work was supported by National Institutes of Health Grant HL 18255. E. L. was supported in part by a Biophysics Fellowship from Cornell University.
REFERENCES 1. Fildes, J. E., Laurence, D. J. R., and Rees, V. H. (1954) Biochem. J. 56, xxxi-xxxii. Stryer, L. (1965) J. Mol. Biol. 13, 482-495. Freedman, R. B., and Radda, G. K. (1969) FEBS Lett. 3, 150-152. 4. Vanderkooi, J., and Martinosi, A. (1969) Arch. Biochem. Biophys. 133, 153-163. 5. Radda, G. K., and Vanderkooi, J. (1972) Eiochim. Eiophys. Acta 265, 509-549. 6. Overath, P., and Trauble, H. (1973) Biochemistry 12, 2625-2634. 7. Trluble, H.. and Overath, P. (1973) Biochim. Biophys. Acta 307, 491-512. 8. Shinitzky, M., and Inbar, M. (1974) J. Mol. Biol. 85, 603-615. 9. Shinitzky, M., and Barenholz, Y. (1974) J. Biol. Chem. 249, 2652-2657. 10. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976)Biochemistry l&4521-4528. 11. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976)Biochemistry 15, 4529-4537. 12. Heyneman, R. A., Bernard, D. M., and Vercauteren, R. E. (1972) J. Chromatogr. 2. 3.
68, 285-288.
13. Gitler, C. (1972) Anal. Biochem. 50, 324-325. 14. Bernard, D. M., and Vercauteren, R. E. (1976) J. Chromatogr. 120, 211-212. 15. Small, D. M., Bourges, M. C., and Dervichian, D. G. (1966) Biochim. Biophys. Acfa 125, 563-580.
16. Bartlett, G. R. (1959)5. Biol. Chem. 234, 466-468. 17. Morrison, W. R. (1964) Anal. Biochem. 7, 218-224. 18. Huang, C., and Charlton. J. P. (1972)Biochem. Biophys. Res. Commun. 46, 1660-1666.