membrane-surfactant interactions micelles labeled · plant physiol. (1974) 54, 527-531...

Plant Physiol. (1974) 54, 527-531

Membrane-surfactant Interactions in Lipid Micelles Labeled with1-Anilino-8-naphthalenesulfonate'

Received for publication December 17, 1973 and in revised form May 13, 1974

GARY M. MILLER AND JUDITH B. ST. JOHN2Agricultural Environmental Quality Institute, Agricultural Research Service, United States Department oJAgriculture, Beltsville, Maryland 20705

ABSTRACT

Sonicated lipid micelles, formed from phospholipids iso-lated from yolks of fresh hen eggs, were used as a model mem-brane system for studying the effects of several surfactants onmembrane properties. The interactions of the surfactants withthe model system were followed through the fluorescence ofthe hydrophobic probe I-anilino-8-naphthalenesulfonate. Thesurfactants investigated were polyoxyethylene sorbitan mono-laurate (Tween 20), polyoxyethylene thioether (Sterox SK),mono-calcium salt of polymerized aryl alkyl sulfonic acids(Daxad 21), and alkylbenzyl quaternary ammonium halide(AHCO DD 50). AU surfactants enhanced fluorescence of themembrane-bound probe, and the degree of this enhancementcorrelated with the previously established phytotoxicity ofthese substances. The results indicate that surfactants canproduce distinct changes in artificial phospholipid mem-branes and suggest that this lipid interaction may accountfor altered membrane permeability characteristics in surfac-tant-treated plants. The effects are observable for surfactantconcentrations as low as 0.0001% (w/v), representing anapproximate 10-fold increase in sensitivity for detecting sur-factant effects compared with previous results on permeabilitychanges in isolated plant cells.

Surfactants are well known for their ability to disrupt thenormal permeability characteristics of plant cells. These sur-factant-induced changes are manifested in a variety of effects,including inhibition of photophosphorylation (15), enhancedgrowth rates (11, 12), and leakage from beet root disks (20)and isolated cells of soybean and wild onion (18). Becausesome of these effects are extremely disruptive to normal cellu-lar development, surfactants also exhibit an inherent phyto-toxicity. A recent investigation in this laboratory has relatedthe phytotoxicity of a number of surfactants to their abilityto alter the membrane permeability of isolated plant cells (18).

Even the relative simplicity of an isolated plant cell repre-sents a complex and, to a large extent, undefined organizationat the molecular level. The most commonly accepted structureof biological membranes involves a biomolecular thickness ofamphipathic lipids, which serves as a fluid supportive phase

' The part of this work represented by Figures 1 and 2 was car-ried out by G. M. M. at the Department of Chemistry, Universityof North Carolina, Chapel Hill, N. C. 27514.

2To whom reprint requests should be addressed.

for the membrane proteins (22). Although the proteins arebelieved to control the transport of substances through themembrane, the lipid bilayer accounts for the permeabilitybarrier and its structure is an important feature of cellulartransport.

The desire for a system that could partially simplify theproblem of lipid-protein interactions has led to the develop-ment of artificial lipid bilayer membranes. Mueller et al. (14)first described and characterized these structures, and recentreview articles illustrate their wide range of applicability (6,8, 21). The preparation and properties of ordered phospholipidvesicles bounded by a single bilayer wall have been describedby Huang (9) and reviewed recently by Papahadjopoulos (16).When dried preparations of naturally occurring phospholipidsare equilibrated with relatively large amounts of water, theyspontaneously form spherical multilamellar structures with thephospholipid molecules in a bilayer configuration, with watertakino up the space between the lamellae and interacting withthe polar groups of phospholipids. Ultrasonic radiation breaksthe large multilamellar particles into smaller ones with fewerlamellae and eventually produces a homogenous populationof vesicles with aqueous inner compartments bounded by asingle bilayer of phospholipids (9, 16). These micelles exhibitmany of the functional properties of biological membranes,especially those related to permeability. The micelles used inthis investigation are considered to be a reasonable model forthe permeability barrier of naturally occurring plant cells.

1-Anilino-8-naphthalenesulfonate is representative of a classof compounds known as fluorescent probes, which show adependence of quantum yield and the wavelength of the emis-sion maximum on the microenvironment of the molecule (1,3). The compounds are virtually nonfluorescent in highly polar(aqueous) solution, but are strongly fluorescent in nonpolarsolvents or when bound to hydrophobic molecules. Stryer (19)has observed an increase of quantum yield and a blue shift inthe emission maximum of ANS' bound to the nonpolar regionsof apohemoglobin and apomoglobin. A similar dependencehas been found in a variety of organic solvents, indicating thatthe polarity of the environment is a major factor in the fluo-rescence of ANS.

This report correlates the fluorescence changes of ANSbound to lipid micelles with surfactant interactions at themembrane surface.

MATERIALS AND METHODS

1, 8-ANS (sodium salt, technical grade) was purchased fromEastman, and was purified by the method of Zingsheim and

'Abbreviation: ANS: 1-anilino-8-naphthalenesulfonate.527

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MILLER AND ST. JOHN

Haydon (24). It was prepared as a 1.8 X 10-2M stock solu-tion in water, and was stored refrigerated and in the dark.

The surfactants were Tween 20 (surfactant No. S-145, poly-oxyethylene sorbitan monolaurate); Sterox SK (No. S-102,polyoxyethylene thioether); Daxad 21 (No. S-064, mono-cal-cium salt of polymerized aryl alkyl sulfonic acids); and AHCODD 50 (No. S-029, alkylbenzyl quaternary ammonium halide)4.Surfactants were obtained from L. L. Jansen, Agricultural Re-search Service, United States Department of Agriculture, andwere dissolved in water on a weight per volume basis. Allsolvents and buffers were analytical reagent grade or higher.Water was distilled and deionized.

Lipids. The lipid fraction used for formation of micelleswas obtained from the yolks of fresh hen eggs, a particularlyrich source of phospholipids. The acetone-insoluble part ofthe homogenized yolks was extracted at 40 C for 1 hour with2:1 (v/v) chloroform-methanol. After the extract was filtered,lipids were precipitated with four volumes of acetone at -20 Cfor several hours. The product was a creamy colored wax,which was stored as a 50 mg/ml stock solution in chloroformat -20 C in the dark. TLC (Silica Gel H, chloroform-metha-nol-water, 65:25:4 v/v/v, rhodamine 6G spray) showed theproduct to be a mixture of phosphatidyl ethanolamine (PE)and phosphatidylcholine (PC), with only a trace of cholesterol.The PE/PC ratio was approximately 2:1. This compositionis similar to the lipid content of many biological membranes.

Preparation of Samples. Lipid micelles were formed from a

0.5 mg/ml solution of lipid in aqueous solution buffered tothe desired pH. Aqueous solutions were buffered with appro-

priate sodium and potassium phosphate salts (5 mM), and thepH was adjusted with HCI or KOH as necessary. A 100-1l(5 mg) aliquot of lipid in chloroform was dried down by a

stream of nitrogen in a small beaker. Ten milliliters of buffersolution were added, and the mixture was sonicated for 3 minat 1.6 to 2.0 amperes on a Branson Model S75 Sonifier. Thesonicated solution was transparent and no lipid precipitatedover a period of several days. Centrifugation at 8000g, 0 C,for 10 min on a Sorval RC2-B centrifuge produced no sedi-mentation. Three milliliters of this solution were transferred toPyrex sample tubes (8 mm X 75 mm). Aliquots of ANS or

the surfactant or both were then added with mixing; and afterthe mixture reached a fluorescence steady state, the fluores-cence was measured on an Aminco Fluoro-microphotometer(American Instrument Co., Silver Spring, Md.). All sampleswere 6 X 10 M ANS and were at 26 1 C. Surfactantswere added in 3-,l volumes to give the desired concentration.Exciting light was provided by an ultraviolet lamp with maxi-mum output at 360 nm and a band-pass filter with peak wave-

length at 360 nm (Corning-751). The fluorescent light was

isolated by a sharp cut-off filter for 415 nm and above (Schott-475). The output from the microphotometer was connectedto an integrator-timer constructed by Aminco. The integrator-timer provided integrated readings over a fixed time scale, witha digital readout. The standardization and units of measure-

ment of the instrumentation will be described under "Results."The shifts of emission maxima were measured on an Aminco-Bowan Spectrofluorometer, exciting at 360 nm. Data illustratedin Figures 1 and 2 were obtained on a Hitachi Perkin-ElmerSpectrofluorimeter equipped with the R-106 photomultiplier.Absorption spectra were taken on a Cary 14 recording spectro-

photometer.

' This information is taken from Jansen et al. (12). Mention of a

trade name or proprietary product does not constitute a guaranteeor warranty of the product by the United States Department ofAgriculture and does not imply its approval to the exclusion ofother products that may also be suitable.

z

I~-

z

wi

wk

'30 270 310 350 390 430 470 510 550 590

WAVELENGTH (nm)

FiG. 1. Absorption (A) and emission (B) spectra of I-anilino-8-naphthalenesulfonate (ANS). A: Concentration of ANS was

3 X 10-1M in absolute ethanol; B: concentration of ANS was 5X101M in absolute ethanol, excitation at 374 nm. Temperature forboth spectra was 25 C.

CONCENTRATION OF ANS x1O 5MFiG. 2. Binding of ANS to sonicated lipid micelles. Micelles

were formed in 0.1 M NaCl as described in "Materials and Meth-ods," 0.5 mg/ml lipid concentration. The pH was 6.0 and the tem-perature was 26 C. Excitation was at 360 nm; the emission was readat the maximum peak amplitude.

RESULTS

The absorption and emission spectra of ANS are shown inFigure 1. When bound to lipid micelles, the probe shows a

strong increase in fluorescence and a blue shift of about 20mm (relative to the unbound ANS in aqueous solution). Fig-ure 2 shows a typical ANS-binding curve for micelles formedin 0.1 M NaCl at pH 6.0. These data illustrate the ANS-bind-ing capacity of the micelles, concentration range for ANS, andshow an ANS saturation of the membrane surface at ANSconcentrations greater than 5 X 10-' M. A double reciprocalplot of these data gave a straight line, from which a slope of2.36 x 10-' M ANS was calculated. This slope may be usedas a relative index of the association constant for ANS bindingto lipid micelles. Because the probe molecule exists as a nega-

tive ion in solution, the extent of binding depends in part on

the ionic state of the lipid polar head groups. This surfaceionization is generally a function of both the surrounding aque-

ous phase and the molecular structure of the lipids in the mem-brane (5, 23). The membrane surface was titrated by varyingthe pH and using the fluorescence intensity of ANS as an indi-cator (Fig. 3). These data indicate that at a pH near 6.0 themembrane surface is at its isoelectric point (neutral point). The

-- . - AB- -

528 Plant Physiol. Vol. 54, 1974



MEMBRANE-SURFACTANT INTERACTIONS

1.0

z

z

z

1V) 0.5

WI

2 4 * 10 12

pH

FIG. 3. Fluorescence intensity of ANS bound to lipid micelles asa function of pH.

Table I. Fluorescenice Chaniges of Micelle-bounzd ANS Iinducedby AHCO DD 50

Mlp+, and AI.+p indicate the intensity change and the order ofaddition of the probe and the surfactant to the micelle solution.The percentage of change is relative to the initial fluorescence inthe absence of DD 50. Experimental details are given in the text.

Surfactant Concn pH | Is+p % Change AIP+s % Change

0.0001 4 0.39 18.8 0.37 17.96 0.40 25.5 0.50 31.87 0.35 19.4 0.43 23.98 0.26 16.0 0.32 19.610 0.19 11.7 0.22 13.5

0.001 6 3.93 3.96

emission intensity of ANS is independent of pH in aqueous so-

lution.Each surfactant that was investigated increased the fluores-

cence of ANS bound to the lipid bilayer. The effect was de-pendent on the surfactant used, the concentration of the sur-

factant, and the pH of the micelle solution. The following re-sults are expressed as a change in fluorescence intensity (Al),using a 10-sec integration period. One unit of fluorescence in-tensity (1.00) is equivalent to the fluorescence intensity of6 X 10o- M ANS in 30% absolute ethanol:water (v/v). Resultswere reproducible to about 0.04 units and were averages of atleast two experiments.AHCO DD 50-Membrane Interactions. The results ob-

tained with AHCO DD 50 are shown in Table I. A markedenhancement of fluorescence was seen at 0.0001% concen-tration and a larger effect was seen at 0.001%. For each con-centration tested, a blank of ANS plus surfactant in the buff-ered aqueous phase was read. At the lowest concentration(0.0001%), AHCO DD 50 increased fluorescence 0.04 in theabsence of any artificial membrane. At 0.001%, the aqueousincrease was 2.98, which is equal to about two-thirds of thereported increase observed for the surfactant-membrane sys-tem.

The extent of membrane interaction, as measured by ANS

emission, was greatest for AHCO DD 50 and reached a maxi-mum at pH 6.0. The effect appeared to be greater when theprobe was added before the surfactant (A10+.). The percentageof change illustrates the relative magnitude of fluorescencechange when compared with ANS alone in lipid micelles (Fig.3). Because of the intrinsic change of ANS-micelle emissionwith pH, Al was judged to be a better measure of the inter-action than was the percentage of change.

Sterox SK-Membrane Interactions. The effect of Sterox SKon the emission intensity was somewhat less than that ofAHCO DD 50 (Table II). Sterox SK exhibited noticeable en-hancement at 0.0001% and an increasing enhancement at0.001% and 0.01%. The maximum increase in intensity againwas at pH 6.0, and the increase was greatest when ANS wasadded before the surfactant at the lowest concentration. Thelatter effect was small, however, and was reversed at the higherconcentrations. In the absence of lipid micelles, the intensityfor the ANS-surfactant-aqueous phase increased only at0.01%. The increase was 0.39 relative to ANS alone in theaqueous phase. A slight clouding was observed on addition ofthe surfactant, which possibly indicates some surfactant micelleformation.

Tween 20-Membrane Interactions. The effect of Tween 20was considerably less than the effect of both AHCO DD 50and Sterox SK (Table III). At 0.0001%, a slight effect was seenat pH 6.0, with no change in intensity at pH 4.0 or 10.0. Thechange was only slightly greater at 0.001%, and 0.01% wasnecessary to produce a sizable change. The maximum en-hancement was again at pH 6.0, and was greater when thesurfactant was added before ANS (AI..+p), in contrast to re-sults with the other surfactants. The change in probe emissionintensity for the aqueous phase was zero at 0.0001% surfac-

Table II. Fluorescenice Chantges of Micelle-boundby Sterox SK

ANS Iniduced

Surfactant Concn pH AI,+P AIP+S

0.0001 4 0.11 0.206 0.29 0.347 0.24 0.328 0.08 0.1410 0.02 0.03

0.001 6 0.56 0.52

0.01 6 1.21 1.16

Table III. Fluorescenice Changes of Micelle-bound ANS Iniducedby Tweent 20

Surfactant Concn pH AIs+p AIp+S

0.0001 6 0.90 0.064,10 -' _1

0.001 6 0.16 0.070.01 4 0.69

6 0.84 0.627 0.538 0.5310 0.78

No change.

Plant Physiol. Vol. 54, 1974 529



MILLER AND ST. JOHN

Table IV. Flutorescenice Chaniges of Micelle-bounld ANS Intdutcedby Daxad 21

Surfactant Concn pH Al p AIPF,

,o

0.0001 4, 6, 7, 10 -' -'0.001 6 0.13 0.150.01 4 0.34

6 0.64 0.677810

0.410.390.30

1 change.

1.25

1.00

LI)

LU

.75

-4 3 2

LOG10 CONCENTRATION

FIG. 4. Surfactant effects on the fluorescence of ANS bound tolipid micelles. A: DD 50; X: Sterox; 0: Tween; *: Daxad. Micelleswere formed at pH 6.0 as described in the text. The surfactant was

added before the probe, and the fluorescence change was recordedas previously described.

tant, 0.05 at 0.001%, and 0.63 at 0.01%. Tween 20 is alsounique in showing an increase in change of intensity (-M) atpH 10.0.

Daxad 21-Membrane Interactions. Daxad 21 at 0.0001%showed essentially no change in the emission intensity of ANSbound to lipid micelles at pH 4.0, 6.0, 7.0, or 10.0 (Table IV).A slight effect was observed at 0.001 %, pH 6.0, and a largeincrease at 0.01%, pH 6.0. The maximum was at pH 6.0, withAlp+, being slightly higher than AL+, A change of intensity(0.21) in the absence of lipid is noticeable only at 0.01%.

Wavelength Shifts in Emission Maximum of ANS in LipidMicelles and in an Aqueous Solution. Because changes inquantum yield of fluorescent probes are usually accompaniedby blue shifts of the emission peak, each of the surfactantswas examined for its ability to produce such a shift. Therewas no wavelength shift for any of the surfactants in the ANSsurfactant-lipid micelle system when we used the surfactantconcentrations that had previously increased emission intensitymost effectively. However, when these same concentrations ofsurfactants were added to ANS in aqueous solution, shiftswere observed for all surfactants except AHCO DD 50. The

shifts were 5 nm for Sterox, 15 nm for Tween 20, and 20 nmfor Daxad.

DISCUSSION

The results of the surfactant-micelle interactions are sum-marized in Figure 4. The order for ANS fluorescence en-hancement is AHCO DD 50 > Sterox SK > Tween 20 >Daxad 21. This order corresponds to that observed in isolatedwild onion cells for the ability of these surfactants to inhibitphotosynthetic14CO2 fixation and for the release of intracellular"4C-material (18). The order is qualitatively similar to the re-sults with "4C-material leakage from surfactant-treated soybeancells and for phytotoxic effects of these surfactants on soybeanand corn, although there was no clear distinction betweenDaxad 21 and Tween 20 in these early tests of Jansen et al(12). In all tests the effects of Daxad 21 and Tween 20 wereconsiderably less than Sterox SK and AHCO DD 50. Our re-sults obtained with lipid micelles are significantly independentof any membrane protein components or cellular metabolicfunctions. Our results provide evidence that the surfactant in-teractions are manifested through the lipid regions of biologicalmembranes. However, they do not rule out the possibility forother types of reactions with metabolizing biological systems.With presently available experimental conditions, and with thegenerally limited knowledge concerning surfactant-membraneinteractions, a definitive explanation of the relative fluorescenceenhancement of ANS by surfactants is not possible.

Radda and Vanderkooi (17) have suggested that observedchanges in ANS fluorescence may arise from any of thesepossible conditions: polarity, accessibility, and orientation con-straint. Various methods, including phase transitions (4), x-raydiffraction (13), and NMR spectroscopy (2) have shown ANSto be located in the polar head region of the lipids forming thebilayer. Similar measurements have shown the probe to besomewhat rigidly constrained and also in contact with thehydrophobic parts of the lipid molecules, resulting in the in-crease of fluorescence and a blue shift in the emission peak.Because the binding of ANS to membranes is strongly de-pendent on the surface charge of the membrane, any suppres-sion or masking of the negative charge from negative phos-phate or carboxylate groups will facilitate the binding of theanionic ANS (5).Haydon and Meyers (7) have suggested that changes in the

surface ionization also may be accompanied by changes inthe hydrophobic core of the membrane. The surfactants sodiumdodecyl sulfate, dodecyl trimethylammonium bromide, anddioctyl lecithin were shown to perturb the nonpolar region ofartificial bilayers by insertion of the hydrocarbon chains ofthe surfactants into that region. Although this interactionnegligibly affected their conclusions concerning membrane po-tentials, our results indicate that surfactant molecules can ap-preciably alter the normal structure of lipid bilayers. Organicanions that inhibit membrane permeability reduce the fluores-cence of ANS with the same order of effectiveness (17). Or-ganic cations have the opposite effect (17), and several undis-sociated organic acids increase the ionic permeability of barleyroots (10).

The importance of surfactant charge is supported by theresults in this paper. AHCO DD 50, a cationic surfactant, in-creased ANS fluorescence most and Daxad 21, an anionic sur-factant, increased it least. Results from Sterox SK and Tween20, nonionic surfactants, were intermediate. All surfactantsshowed a maximum effect at pH 6.0, the "neutral point" ofthe membrane surface as determined from Figure 3. Thesedata would seem to indicate that the nonpolar parts of thesurfactants are most influential in binding to the membranes.

Plant Physiol. Vol. 54, 1974530



MEMBRANE-SURFACTANT INTERACTIONS

The enhancement of ANS emission by surfactants in aque-ous solution is believed to be the result of micelle formationfrom the surfactants. The aqueous enhancement is low or non-existent for low concentrations, but is strong in all tests forthe 0.01% concentrations. The results concerning wavelengthshifts provide additional information on the surfactant modeof action. The degree of shift observed for each surfactantmay indicate the ease of micelle formation by these compoundsbecause no peak shift occurs at low concentrations (probablybelow critical micelle concentration).

Because the wavelength of ANS emission is shifted in eithera solution containing ANS and surfactant or a solution contain-ing ANS and lipid micelles, a more pronounced shift couldoccur in a system containing ANS, surfactant, and lipid mi-celles. A three-component system might be expected to shiftthe ANS emission maximum to a greater extent due to the in-creased importance of one or more of the following parame-ters, polarity, accessibility, and orientation constraint, whichaffect the fluorescence of ANS. Even at high concentrations ofsurfactants the shift did not exceed that attributable to theANS-lipid micelle interaction. One or more of the followingalternatives are suggested: (a) the shift in ANS fluorescencecaused by the surfactant in the lipid micelle phase is equivalentto that caused by the lipid and hence is undetectable as aseparate entity; (b) the surfactants tested can form micelles inthe aqueous phase, but not in the lipid solution; (c) the shiftby the surfactant in lipid micelle solution is different from theshift observed in water and is negligibly small when comparedwith the shift for the lipid (about 20 nm).

The primary factor contributing to the increase of ANSemission in surfactant-treated lipid micelles is believed to bean alteration in membrane structure that increases the per-meability of the membrane to a variety of molecules, includingANS. Interaction of the surfactants at the membrane surfacecould also conceivably alter the environment of ANS in severalrespects. The probe may interact with the hydrophobic regionsof the surfactant and the motion of ANS may be constrainedas a result of a tighter packing in its binding region. Becauseof the limitations on the experimental system as discussed byRadda and Vanderkooi (17), the separation of these effectsinto those produced by changes in polarity, accessibility, andorientation constraint is difficult at this time. Consequently, theincrease in relative fluorescence of ANS, bound to lipid mi-celles, in the presence of surfactants cannot be adequately ex-plained. Most likely polarity, accessibility, and orientation con-straint all undergo some degree of change as the surfactant isbound near the polar-nonpolar interface in the bilayer.

The results provide further evidence that these surfactantsare able to alter the lipid portions of membranes. The altera-tions may explain many of the observed properties of thesurfactants in growing plants, including the phytotoxicity re-sulting from a disruption of normal membrane structure andactivity. The model membrane system is a valuable tool forthe investigation of problems related to plant membrane struc-ture and function.

Acknowledgments-We are grateful to L. L. Jansen for providing the sur-factants and to R. R. Little for skilled technical assistance.

LITERATURE CITED

1. CHANCE, B., C. LEE, AND J. K. BLASIE, eds. 1971. Probes of Structure andFunction of Macromolecules and Membranes, Vol. 1. Academic Press, NewYork.

2. COLLEY, C. M. AND J. C. METCALFE. 1972. The localization of small moleculesin lipid bilayers. FEBS Lett. 24: 241-246.

3. EDELMAN, G. M. AND W. 0. MCCLURE. 1968. Fluorescent probes and theconformation of proteins. Acc. Chem. Res. 1 (3) :65.

4. FAUCON, J. F. AND C. LUSSAN. 1973. Aliphatic chain transitions of phospholipidvesicles and phospholipid dispersions determined by polarization of fluo-rescene. Biochim. Biophys. Acta 307: 459-466.

5. FLANAGAN, M. L. AND L. R. HESKETH. 1973. Electrostatic interactions in thebinding of fluorescent probes to lipid membranes. Biochim. Biophys. Acta298: 535-545.

6. GOLDUP, A., S. OsiKI, AND J. F. DANIELLI. 1970. Black lipid films. In: J. F.Danielli, M. D. Rosenberg, and D. A. Cadenhead, eds., Progress in Surfaceand Membrane Science, Academic Press, New York. pp. 193-260.

7. HAYDON, D. A. AND V. B. MEYERS. 1973. Surface charge, surface dipoles, andmembrane conductance. Biochim. Biophys. Acta 307: 429-443.

8. HOWARD, R. E. AND R. M. BURTON. 1968. Thin lipid membranes with aqueousinterfaces: apparatus designs and methods of study. J. Amer. Oil Chem.Soc. 45: 202-227.

9. HUAN-G, C. 1969. Studies on phosphatidylcholine vesicles. Formation andphysical characteristics. Biochemistry 8: 344.

10. JACKSON, P. C. AND J. M. TAYLOR. 1970. Effects of organic acids on ion uptakeand retention in barley roots. Plant Physiol. 46: 538-542.

11. JACKSON, W. T. 1962. Use of carbowaxes (polyethylene glycols) as osmoticagents. Plant Physiol. 37: 513-519.

12. JANSEN-, L. L., W. A. GENTNER, AND W. C. SHAW. 1961. Effects of surfactantson the herbicidal activity of several herbicides in aqueous spray systems.Weeds 9: 381-405.

13. LESSLAUER, W., J. E. CAIN, AND J. K. BLASIE. 1972. X-ray diffraction studiesof lecithin bimolecular leaflets with incorporated fluorescent probes. Proc.Nat. Acad. Sci. U.S.A. 69: 149941503.

14. MUELLER, P., D. 0. RUDI-N, H. T. TIEN, AND W. C. WESCOTT. 1963. MNethodsfor the formation of bimolecular lipid membranes in aqueous solution. J.Phys. Chem. 67: 534.

15. NEUMANN-, J. AND A. JAGENDORF. 1965. Uncoupling photophosphorylation bydetergents. Biochim. Biophys. Acta 109: 382-389.

16. PAPAHADJOPOULOS, D. 1973. Phospholipids as model membranes: monolayers.bilayers, and vesicles. In: G. B. Ansell, J. N. Hawthorne, and R. M. C.Dawson, eds., Form and Function of Phospholipids. American Elsevier,New York. pp. 143-169.

17. RADDA, G. K. AND J. VANDERKOOI. 1972. Can fluorescent probes tell us any-thing about membranes? Biochim. Biophys. Acta 265: 509-549.

18. ST. JOHN-, J. B., P. G. BARTELS, AND J. L. HILTON. 1974. Surfactant effects onisolated plant cells. Weed Sci. 22: 233-237.

19. STRYER, L. 1965. The interaction of a naphthalene dye with apomyoglobin andapohemoglobin-a fluorescent probe of nonpolar binding sites. J. Mol. Biol.13: 482-495.

20. SUTTON-, D. L. AND C. L. Foy. 1971. Effect of diquat and several surfactantson membrane permeability in red beet root tissue. Bot. Gaz. 132: 299-304.

21. TIEN, H. Ti A-D R. E. HOWARD. 1972. Bimolecular lipid membranes. In: R. J.Good, R. R. Stromberg, and R. L. Patrick, eds., Techniques of Surface andColloid Chemistry and Physica. Marcel Dekker, New York. pp. 109-211.

22. VAN-DERKOOI, G. 1972. Molecular architecture of biological membranes. AnnN. Y. Acad. Sci. 195: 615.

23. VANDERKOOI, J. AND A. MARTINOSI. 1969. Use of 8-anilino-1-naphthalenesulfonate as a conformational probe on biological membranes. Arch. Bio-chem. Biophys. 133: 153-163.

24. ZINGSHEIM, H. P. AND D. A. HAYDON. 1973. Fluorescence spectroscopy ofplanar black lipid membranes-probe adsorption and quantum yield deter-mination. Biochim. Biophys. Acta 298: 755-768.

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