A temperature gradient method for lipid phase diagram construction using time resolved x ray diffraction

A TEMPERATURE

GRADIENT

METHOD

FOR

LIPID

PHASE

DIAGRAM CONSTRUCTION

USING

TIME-RESOLVED

X-RAY

DIFFRACTION

MARTIN CAFFREYAND FREDERICK S. HING

Section of Biochemistry,Molecular and Cell Biology, CornellUniversity,Ithaca,NewYork14853

ABSTRACT Amethod that enables temperature-composition phasediagram construction atunprecedented rates is described andevaluated. The methodinvolvesestablishingaknowntemperaturegradient along the length ofametal

rod.Samples of different compositions contained in long,thin-walled capillariesarepositioned lengthwiseontherod

and"equilibrated" such that thetemperaturegradient iscommunicated into the sample. The sample is then moved throughafocused,monochromaticsynchroton-derivedx-raybeam and theimage-intensified diffractionpatternfrom thesampleis recorded onvideotapecontinuously in live-time asa function of position and, thus, temperature. The

temperature at which the diffraction patternchanges corresponds to a phase boundary, and the phase(s) existing

(coexisting) oneither side ofthe boundary can be identified on the basis of the diffraction pattern. Repeating the measurementonsamplescovering the entire compositionrangecompletesthe phasediagram.Theseadditionalsamples canbeconveniently placedatdifferentlocationsaroundthe perimeterofthecylindrical rod and rotated into position for diffraction measurement.Temperature-composition phasediagrams forthefully hydrated binary mixtures, dimyris-toylphosphatidylcholine(DMPC)/dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE)/DPPC, have been constructed using thenewtemperaturegradient method.Theyagreewellwith and extend

the results obtained by other techniques. In the DPPE/DPPC system structural parameters as a function of temperatureinthe various phases including thesubgel phasearereported. The potential limitations ofthissteady-state

methodarediscussed.

INTRODUCTION

An understanding of lipid-lipid and lipid-water interac-tions and how these depend on the chemical properties of thelipids requires a knowledge of the conditions governing mesomorphic phase formation. This information is neatly summarized in a temperature-composition phase diagram. Anumberof different methods have been used to construct phase diagrams of lipid andlipid/water mixtures, includ-ing calorimetry, electron microscopy, light scatterinclud-ing, nuclear magnetic and electron spin resonance (ESR), infra-red, fluorescence and Raman spectroscopies, and x-rayand neutron diffraction. Of these, x-ray diffraction is particularly useful because of its ability to identify phases onbothsidesof a phase boundary and to localize the phase boundary itself. Incontrast,other methods can detect the phase boundary but provide little unique information concerning phase identity. Using conventional x-ray sources,phasediagram construction by x-ray diffraction is alaborious andtime-consuming process due to low photon flux and consequent lengthy exposure times. With the advent ofsynchrotron radiation and its greatly enhanced x-ray flux, a large amount of diffraction information can be collected in a relatively short perod of time, allowing phase and structural information to be obtained at signifi-cantly increased rates. Seeking to take advantage ofthis

fast datacollection, wedevelopeda new method forphase diagram construction using fully hydrated mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoyl-phosphatidylethanolamine(DPPE)andmixtures of DPPC and dimyristoylphosphatidylcholine (DMPC). The method makes useoftime-resolved x-ray diffraction

mea-surements (Caffrey and Bilderback, 1983, 1984; Caffrey, 1984, 1985)onlipidsamples contained incapillaries along which a temperature gradient has been established. The essential features of the methodareillustrated inFig. 1 for a binary lipid mixture. Imposing a temperature gradient along the lengthof thesamplecapillarycan be viewed as

positioning the capillary on a line of constant overall composition (isopleth) in thephase diagram. In this way, thedifferentphasesandphase boundariesarelocalizedat

different points along the length of the sample capillary. By moving the capillary through the x-ray beam while continuously recording the two-dimensional diffraction pattern, phase changes may be detected by changes in diffraction and the pattern itself serves to identify the phase(s) present at a particular temperature.

Repeating

the measurement on samples of different

composition

X-RAY

T2

r

L-L+S

s .b

.a...

T Pure A

Composition

FIGURE 1 Principle of the temperature gradient method for phase diagramconstruction illustratedusingabinarymixture oflipidsAand B. The system exhibitsasolidorgel(S),aliquid-crystalline phase (L),anda regionofphasecoexistence(S+ L).Acapillary containingthelipidor lipid mixture offixedoverallcompositionisplacedonthe temperature

gradient. Dependingonsample compositionandtheparticular gradient

imposed,variousphasesformalongthelengthof thecapillary, whichare

subsequentlylocalized, identified,andquantified bytime-resolvedx-ray

diffraction. The origin of a concentration gradient within the phase coexistence regionis accountedforasfollows. A temperaturegradient from T, to T2 is established along the length ofthe capillary, which containsasamplewhose overallcompositionisequimolarinlipidAand B.Thegiven phase diagram impliesthat thecompositionof the Lportion ofthesampleis 0.4 molfraction Batpointb and 0.3 molfractionBat

pointa.Consequently,there isadecreasingconcentrationgradientin B

from point bto pointa and a gradient ofopposite sense forA in the coexistenceregion.

patternsarerecorded alsomeansthat

problems

that

might

arise

by

other

techniques

due to spontaneous

sample

orientationareobviated in the present method.

The

phase properties

of

fully hydrated

mixtures of DMPC and DPPC and of DPPC and DPPEwerestudied

using

the

temperature-gradient

method and

compared

with results in the literature obtained using a variety of other

physical techniques.

Thetwosets areshowntobe in excellent agreement. The

temperature-gradient

data extendour

knowledge

of the

low-temperature region

of the

DPPC/DPPE phase diagram

and

provide

structural infor-mation on the various

phases

as a function of

tempera-ture.

MATERIALS AND METHODS

Materials

L-a-DMPC and L-a-DPPC werepurchased from Avanti PolarLipids,

Inc. (Birmingham, AL). L-a-DPPE was obtained from Calbiochem-Behring Corp.(La Jolla, CA). Using 100-,g samples, lipidswerejudged to be _98% pure by thin-layer chromatography in acidic and neutral solventsontwosolid supports(K5Fsilicagel,WhatmanInc.,Clifton,NJ; Adsorbosil5-Pplates, AppliedScienceLaboratories, Waltham, MA)as

previously described (Caffrey andFeigenson, 1981). Lipids were used without further purification. All other chemicals and solvents wereof reagent grade(Caffrey, 1985).

Sample Preparation

Lipids were dissolved in eitherCHC13 (DMPC andDPPC) or aCHC13/

MeOH (9/1, vol/vol) solution (DPPE). To prepare lipid mixtures,

appropriateamountsof eachlipid in organic solvent were added (30-50 mgtotal)to aglasstesttube and the solvent evaporated under argon gas.

Completesolvent removal fromthethinly shelled lipid film was accom-plished under high vacuum for at least 12 h. The dried lipid was suspended in either Milli-Q purified water (Millipore Corp., Bedford,

MA) for DMPC/DPPC mixtures or 100 mM HEPES, pH 7.0 for DPPC/DPPE mixtures, to a final lipid concentration of -1 mg/ml,by alternately incubatingat700C andvortexing until a creamy,

homoge-neous suspension was obtained. Samples were transferred to 1.5-ml

centrifugetubes and spun for 5min in anEppendorf centrifuge. Excess clear supernatant was removed and mixtures were pipetted into

1-mm-diamquartz x-raycapillaries (Charles Supper Co., Natick, MA) and spundownattopspeedinaclinicalcentrifuge (Model 1528E; Interna-tionalEquipment Co.,Boston, MA). Because of the high viscosity of the

hydrated gel phase lipids,someof themixturesrequiredheating above the

geltoliquid-crystaltransition temperatureduring centrifugationto effect

pelletingin thecapillaries.Inpreparingsomeparticularlythick mixtures,

a number ofcycles of pipetting lipid into the sample capillary and

centrifuging while heating were required. Capillaries were filled to

providealipid sample4cmlong,flame-sealed, and furthersecured with 5 min epoxyappliedtotheflame-sealedend.DPPC/DPPEmixtures were storedat40C for 3-5 d beforetakingx-ray measurements. The DPPC/ DPPEsamplesweretransferredtothegradientrodfromcoldstorage as

quicklyaspossible (-4 s)toavoidloss ofanysubgelphase(seebelow) thatmighthave formed. Allsampleswereincubatedonthegradient rod

atleast 15min beforetakingmeasurements.Thin-layerchromatography

(TLC)ofsamplesafterdiffractionmeasurementsshowed _98%purity of thelipid.

Temperature

GradientApparatus

A schematic view of thetemperaturegradientapparatus is shown in Fig. 2.Itconsists ofastainless steelcylindricalrod(12cmlongand 1.6cmin

diameter)withasmall heater(model 8332;HotwattInc., Danvers,MA) connectedto avariablevoltagesource(The SuperiorElectricCo.,Bristol,

CT) positioned in one end. Temperature control at the other end is

provided byacirculatingwaterbath. Copper-constantanthermocouples

(0.015indiameter, Omega Engineering Inc., Stamford, CT)areplaced

alongthelengthofthe rod in thepositionsshown inFig.2tomonitor the

imposed temperature gradient. Thermocouples were periodically cali-brated inanice/waterslush. Thesample capillaryisplacedontop of the rodandsecured withCrycongrease topromotegood thermalcontact.

The roditselfis attachedto agoniometerhead mountedon a

computer-controlled,motorizedX-Ytranslation stage. This arrangementprovides precise positioning ofthesampleinthe beam.Duringascan,horizontal stageposition was monitored continuouslywith a linearpotentiometer (model245103, RVT-S128A, 500, 1163A; Link Aviation Inc.,Acton,

MA)whose outputwasrecorded and correlated with x-ray beamposition along the capillary and with sample temperature. Position along the

sample capillaryand thussampletemperaturearerecordedonvideotape simultaneouslywith the x-ray diffraction patternbydirectingthe linear

potentiometeroutputto adigitalvoltmeterinterfaced withacharacter generator (serial No. S92, 131-55-11234; Chrono-Log Corp., Haver-town, PA). The generator's output is inturn routed to the videotape recorder.

The temperaturegradients establishedonthe rod atdifferentwater

bath and heater (Variac) settings were measured and shown to be

approximatelylinearoverthe central 5.5-cmlengthof therod,andare

circulatinng

waterboth x-ray_beam

ster

FIGURE 2 Schematic view ofthetemperaturegradientapparatusinthe colt revolver arrangement.Temperaturecontrols at the two endsof

the rod (heater andwater bath) maintain the temperature gradient.Thermocouples monitortemperaturealongthe rod. The eucentric

goniometerhead allowspreciseadjustmentof thecapillariesinthex-raybeam. Horizontaltravel of thesample capillarythroughthe beamis

providedbythex-ytranslation stage. In theupperportionof thisfiguretheoriginofatemperaturegradienttransverse tothecapillary long

axis isexplained.Thecapillaryshown containsalipidwithL;andL.phaseswithatransitiontemperatureof ti. Att.thelipid adjacenttothe

gradient rod undergoes the transition. Becauseof thetransversetemperaturegradientinthecapillaryand becauset,>roomtemperature, the

phaseboundary is shifted towardtherightasdistancefromthe rod increases.

remarkably stable. Forexample, fora35-900C gradient, thetemperature

varied by±IOCat86.OOC and ±0.25°Cat40.30Cover a2-hperiod.

The thermocouples aresecured in holes drilled in the rod and the

readings reflect thetemperaturenearthecenterof the rod.However,the

sample capillary sits on the rod surface and to get direct sample

temperaturereadingsit isimportantthat there benosignificant

differ-encesbetween theinternal andexternal temperaturesalongthelengthof

the rod. Thiswasconfirmedbyindependentmeasurements.

X-ray Diffraction

The time-resolved x-ray diffraction method and apparatus has been

previously described (Caffrey and Bilderback, 1983, 1984; Caffrey, 1985).Focused, monochromaticradiationat1.56 A from the Al lineat

the CornellHigh Energy SynchrotronSource(CHESS)wasusedasthe

x-ray source during which time the Cornell Electron Storage Ring (CESR) was operated at 5.2 GeV and 20-30 mA of electron beam

current. Allmeasurements weremade witha 0.3-mm-diam collimator

(Charles Supper Co.). Diffraction patternsweredetectedusinga modi-fied three-stage imageintensifier tube (model 1267-1; Varo Inc.,

Gar-land, TX) andaCohu 500 series TVcamera(model 5272-2220/AL4;

Cohu Inc., San Diego, CA) and were recorded on a U-Vision NV-9240XDvideocassetterecorder(Panasonic Co., Secaucus, NJ). Calibra-tion of theintensifier/camerasystemwasaccomplished usingastandard fully hydratedDPPC multilamellersampleat250C andusing the 64-A first-orderlamellar reflectionatlow-angles and the 4.2-A rigid acyl chain

reflectionatwide-angles.

X-ray capillarieswerepositionedontopof thegradient rodasshownin

Fig.2andCrycongrease wasusedtopromotegood thermalcontact.This grease consists of copper filings in a grease base. The grease has a wide-angle scattering profile similar tothat ofthe "fluid" lipid phase. Throughout thesemeasurementscare wastakentoensurethat thegrease

didnotcontributetotherecordeddiffractionpatterns.

Foraccurated-spacingmeasurementsas afunction oftemperature,the sample capillary mustremain atafixedsample-to-detectordistanceas

thesampleismovedthroughthex-raybeam. Thiswasassuredbyproper alignment of the goniometer head and was verified by viewing the

capillary throughanoverheadmicroscope fittedwitheyepiececrosshairs

whilemoving thestageback and forth andby measuring the interlamellar

(room temperature)

x-ray

capillary

phase

boundary

.

LL

~x-ray

'

gradient

rod beam

d-spacing of fullyhydrated DPPC at 200C as a function of position on the

rod. The d-spacings vary by ±0.5 A, indicating a constant

sample-to-detectordistance along the length (4 cm) of the capillary.

The sampleislevelled using the goniometer so that the x-ray beam

travels through themiddleofthe sample capillary, maximizingdiffracted intensity and ensuring that diffracted intensity is comparable at all points in the sample. This is complicated, however, by the fact that the capillaries are not of uniform diameter along their length, and by inhomogeneities in the sample (seebelow).

In certain instances sample length was not sufficient to cover the temperature gradient rangeofinterest. In such cases the sample was progressively moved to higher temperatures on the rod, and measure-ments weretaken sothat theentire temperature range could be examined. Failure to make measurements over continuous temperature ranges

resultedin missing datain oneofthed-spacing plots (Fig. 5).

Tomaximize resolutionlow-and wide-anglediffraction regions were

examined inseparatescans.Diffraction patterns were recorded

continu-ously fromthe low to the high temperature end ofthe rod at a rate

(dependingonthe steepnessofthegradient) corresponding to-0.7C°/s.

Data

Analysis

Thedata consist of two-dimensional diffraction patternsfrom thelipid

samples recorded onvideotapeas afunction oftime and temperature.

Dataanalysis to determine phase transition temperatures and structural parameters wasperformedin three ways. First,thetemperaturegradient scanswerevieweddirectlyon atelevision monitoratboth low-angleand

wide-angle and any noticeable changes in the diffracted intensity or peak

position during thescan werenoted.Second,one-dimensional diffracted intensity line scans through the vertical diameter of the diffraction patterns wereobtainedusing an image processor (Grinnell Image Display

Buffer, model GMR-274; Grinnell Systems Corp., San Jose, CA) as

previously described (CaffreyandBilderback,1983). Plotting the data in

compositeformgreatlyfacilitates detectionofchanges in intensity and/or

peakposition (Figs.3and 4). Finally,d-spacingsforthe lipid samples as a

function oftemperature werecalculated from the line scans. A phase transition isindicated in thecorrespondingd-spacing plot (Fig. 5) by a

suddenchangein the repeatperiod withachange in temperature.

RESULTS

DPPC/DPPE

Mixtures

Thelong-spacingsoffully hydratedDPPC/DPPE multila-mellarmixturesas afunction of temperature on a10-700C gradient are shown in Fig. 5. Pure DPPC exhibited changes with temperature much like thoseseenby Janiak et al. (1976) and by Inoko and Mitsui (1978) for the formation by P,B phase at 34.5° and 35°C, respectively, using both differential scanning calorimetry (DSC) and x-raydiffraction.Alarge increase in thed-spacingfrom65 to 72 A occurred at 350C. In the wide-angle region, the broad shoulder at -4.06A (220C) onthe wide-angleside of the "rigid" acyl chain reflection at4.2 A appeared to move inward and becomesymmetric with the

4.2-A

line; however, this changewas moredifficulttodiscernbecause of interference by the adjacent diffuse water peak (data not shown). Similarly, at 0.1 mol fraction DPPE, forma-tion of the

Pp

phase was seen as a large increase in the d-spacingof thelipidfrom 67to74Ain the 39.50to440C temperature range accompanied by the same changes in the wide-angle region as was seen in pure DPPC. For mixtures with 0.2 mol fraction DPPE andabove, no such

I

L

ApNmmo

h

.Liv

~~ - ~ -~~---

--63A

58.0"C

57.4-C

156.8"C

55.4-C

55.0.C

mm

54.6boC

54.0"C

6mmad

53.6"C

52.96C

mm

0 0.00a 0.0016

SinO/x,

(A)-FIGURE 3 Changes inlow-anglex-raydiffractionattheorder-disorder

transition for fully hydrated DPPC/DPPE (0.4/0.6, mol/mol).

Diffrac-tionpatternswererecordedonvideotape and image processedtoobtain diffracted intensity profilesasdescribed under Materialsand Methods.

Eachprofilerepresents theaverageof nine videoframes (300msat30

frames/s). Major peaks are due to first-order lamellar reflections. Second-order reflectionscanbeseenatthe lowertemperatures.The low temperaturefirst-order peak correspondsto ad-spacing of70A.

increase in the lamellar repeat norrearrangements in the wide-angle occurred over the temperature range

120

to 700C.

At DPPEmolefractionsof <0.3, evidence for the subgel phase(Chenetal., 1980; Ruoccoand Shipley, 1982)atlow temperatures was indicated by a smaler lamellar repeat and/or by additional reflections in thediffraction pattern atintermediateangles(Fig.6).In pure DPPCanincrease ind-spacing from 62 to 64 Aoccurred from 120 to 160C, and in the mixturecontaining 0.1 mol fraction DPPE an increase from 64.8 to 67

A

occurred over a temperature range of 110to 140C. Also,an additionalpeakat -3.85A characteristic of thesubgel phase was seen for thesetwo samples, which gradually moved to lower angles and finally merged with the sharp

4.40-A

reflection athigher temperatures. This coincided with thedisappearanceof the

r

dmd,AN-

..

-- EftwWommmmmom

a

A

_ I --A

in,

vA.

No

9

r

4.6A

4.2A

o.1o0 0.119

SinO/x,

(A)Y'

FIGURE 4 Changes in wide-angle x-ray diffraction at the

order-disordertransition for fully hydrated DPPC/DPPE (0.4/0.6, mol/mol). Foradditional detailsseelegend to Fig. 3. Profiles represent the average

of20videoframes(670 ms at 30frames/s).

intermediate-angle reflection at -6.7

A

seen at low tem-perature. For DPPC/DPPE mixtures containing 0.2 and 0.3molfraction DPPE, the lamellar repeat at low temper-ature(<250C) increased much less, although their rates of increase still appeared discontinuous. An additional peak to the wide-angle side of the

4.4-A

reflection did appear broader and more intense at lower temperatures, and reflections at intermediate angles were noted in the diffrac-tion patterns recorded on videotape, which were absent at highertemperatures (>200C). These reflections were gen-erally neither as intense nor as well-defined as those seen for pure DPPC and the 0.1 mol fraction DPPE mixture possibly reflecting a less ordered subgel phase in the 0.2 and0.3molfractionDPPEsamples. Longer incubations at 40C may be required to fully develop the subgel phase in these higher DPPE samples. The temperatures at which these intermediate reflections completely disappeared agreed well with thecompletion of the slight increase in d-spacing seen in the d-spacing plots (Figs. 5 and 7).

Additional reflections at intermediate angles in the diffraction pattern were observed for pure DPPE at low temperatures, suggesting the existence ofa subgel phase under these conditions. These reflections faded gradually astemperatureincreased until -240C, when they couldno

longer be detected andafully developed lamellar gelphase emerges. There was no corresponding increase in the lamellarrepeat overthistemperature range,however. This result differs from the observations of Wilkinson and Nagle (1984) who noted in the case of DMPE a direct transition from the subgel to a liquid-crystalline phase without an intermediate L phase. The lamellargelphase of DPPE,therefore, doesnot appear tobeunduly metasta-ble as it was still seen in the 240 to 630C range after 5 d incubation at 40C, and there was no increase in the corresponding order-disorder transition temperature (630C). In lipid mixtures containing between 0.4 and 0.8 mol fractionDPPE,nodiscontinuityin the rate of increase of thelong-spacing at lowtemperatures was observed nor werethereadditional reflections at intermediate angles in the diffraction pattern, which might suggest the presence of asubgel phase. These mixtures either do not form this latterphase or they require longerthan 3-5 d at 40C todo

so.

The order-disorder transition was apparent in the dif-fraction patterns as the disappearance of the sharp rigid acyl chain reflection at -4.2 A and the emergence of a diffusebandcentered at 4.6 A due to "fluid" acyl chains. In the low-angle region of the diffraction pattern, an additional sharpline became visible to thewide-angle side oftheoriginal lamellar reflectionastemperatureincreased throughthetransition, indicatingcoexistence of the lamel-lar gel and liquid-crystalline phases (Caffrey and Bilder-back, 1983; Caffrey, 1985). The reflection due to the liquid-crystalline phase gradually increased in intensity andeventually replaced that duetothe lamellargel phase at the higher temperature. These changes are shown in Figs. 3to 5fora DPPC/DPPE (0.4/0.6) sample.

The temperatures of the various phase transitionsover theentire range ofDPPC/DPPEratiosareplottedinFig. 7along with theproposedtemperature-compositionphase diagram. There issome uncertainty as towhether the gel phases (LaorL4,

Pp

or

Pp)

inthe mixedlipid rangeareof thef3 or,B' type since pure DPPC has itsacyl chainstilted with respect to the bilayer normal (fi') while DPPEdoes not (p3, McIntosh, 1980; Hauser et al., 1981). Because careful static measurements were not performed in the intermediate DPPC/DPPE composition region,a determi-nation of chain tiltanglewas notmade in the presentstudy. Consequently, the phase fields below the lamellar liquid crystalline and above the subgel phasearedesignated

Lgei

andPgeI inFig.7.

DMPC/DPPC

Mixtures

0.3

0.2

.

0.1

ddp~~~~~~~.

* _ .

.9

w

10.0- 20.0 30.0 40.0 50.0 60.0 70.0 80.0 TEMPERATURE (C)

0

o

co

a

0

0

*0to to

0

0

CO

la

0

rw

o

a

Ca

0

o

0

0co

O 0.6

o°.

~ ~ ~ ~ ~ ~ ~ .

N~~~~~N

o

- 905

0.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 TEMPERATURE (QC)

FIGURE 5 Longspacings of fully hydrated DPPC/DPPE mixtures calculated from linescansof first-order lamellar reflections recorded

using the time-resolvedx-raydiffractionmethod. The mole fraction DPPE ineach mixture is indicated.Duetosample lengths of<4cm,

samples containing 0.7, 0.8, and 1.0 mol fraction DPPEweremovedtosuccessively highertemperaturesand scanned again until theentire

temperaturerangewascovered.

200to400Cis shown inFig.8. The transitiontemperatures

correspond to onset andcompletion ofthe order-disorder transition andwereobtainedsolelyfromviewingthe video recorded wide-angle diffraction patterns as a function of temperature. Thechanges seenin the diffractionpatterns

atthe various transition temperaturesweresimilar to those described above for mixtures of DPPCandDPPE.

The phase diagram for the DMPC/DPPC system obtained using the temperature-gradient method agrees

wellwiththatobtained byother methods (Shimshickand

McConnell, 1973; Chapman et al., 1974; Luna and

McConnell, 1978). Asnoted for theDPPC/DPPEsystem

(see below), the region of coexistence obtained using the

temperature-gradient method is generally narrower than that obtainedbyothermethodsalthoughitstill falls within thesametemperaturerangeforagiven composition.

DISCUSSION

The temperature gradient method has been used to con-struct temperature-composition phase diagrams for fully hydrated DPPC/DPPEand DPPC/DMPCmultilamellar

mixtures. In both instances our findings agree with and extend data obtained by a variety of other methods. A

phase diagram forDPPC/DPPEinexcesswaterhas been

proposed by Blume et al. (1982 and references therein)

basedon13Cand 2H-NMR studies andondata fromESR, DSC, chlorophyll a fluorescence, and Raman spectrosco-py. The phase coexistence regions as determined by the present temperature gradient method are generally nar-rowerthan thosereported for other methods. Thismaybe due to a lower sensitivity of x-ray diffraction to subtle

changes in molecular arrangements (MelchiorandSteim,

oN~~~E

:1 0.8

0

a

0 N

0 30 0

0

1.

0 0

0

0

CO

o 0 C

N

.0

0

zo

0

o

o co

0

N

0

0

id

0 0

03 0

0k

pco

N r

0

%.Oo

0

a

2i

0

%%S

3

IJJ

1

o

0 1 2

I

17

3

4

0

0

c0_U)

C.)

I-:

L&J

a-LLI

I--0 0

0

0

i

fn

0

.0

CS

0.1 02 0.3

2(Sino)iA,

AK1

FIGURE 6 Microdensitometric scan of an x-ray photograph of the

subgel phase in fully hydrated DPPC multilamellar vesicles. Samples wereincubated at 0-40C for 13 d before measurement at 80C in a 1-mm quartzcapillary using a 0.3-mm collimator. Exposure time was 15 min at 5.24GeV, 15 mA. The d-spacings of the peaks indicated are as follows: 1, 5.89; 2,2.95; 3, 1.96; 4, 1.47; 5, 1.19; 6,0.990; 7,0.930; 8,0.824; 9,0.668; 10, 0.646; 11,0.601; 12, 0.540; 13, 0.495; 14,0.485, 15, 0.460; 16, 0.451; 17, 0.440; 18, 0.418; 19,0.385 and 20, 0.355 nm. The scan was made

using a microdensitometer (model 11Ic; Joyce-Loebl, Gateshead,

England.)

1979). Itmayalsoindicate that thetransitions themselves are intrinsically more sharp and that the temperature gradient method accurately reflects this. In the current work, the

Pg.,

phase was seen at <0.2 mol fraction DPPE as opposedtoconcentrations of upto0.5mol fraction DPPE observed by Blume et al. (1982). This difference is partially attributable to the different techniques used inasmuch as the temperature gradient method is not an equilibrium method (see below). The phase diagram pro-posed in Fig. 7 is similar tothat described by Luna and McConnell (1978) for fully hydrated DPPE/DMPC mix-tures in having three two-phase regions which meet at a three-phase linelocated, in the presentcase, at460C.

Transition temperatures determined from d-spacing plots (Fig. 5) and from visual inspection of diffraction patternsrecorded onvideotapecompared well (+1°C)with

each other. Further, the low-angle diffraction data were moresensitive tothe startoftheorder-disorder transition than thewide-angle data,since theappearance ofasharp reflection at low-angles was easier to detect than an increase in diffuse scatter at wide angles (Caffrey and Feigenson, 1984). However, the sensitivity for detecting completion of the transitionwas better atwide angles. This

0.00 0.20 0.40 0.60 0.80

XDPPE in DPPC

1.00

FIGURE 7 Proposed phasediagramfortheDPPC/DPPE multilamellar

system inexcessaqueousphase. Onset andcompletion phase transition

temperatures weredetermined bythreedifferenttypesof observations of

diffraction data: (open circle) d-spacing plots; (open square) visual inspection of low-angle diffraction data on a video monitor; (open triangle) visual inspection of wide-angle diffraction data on a video

monitor. Solid andopensymbols denote,respectively,onsetand

comple-tion transitions in the direction of increasing temperature. Estimated

errorin temperature is±1.50C.It isemphasizedthatalong eachisopleth phase determination hasbeenmadecontinuouslyoverthe full

tempera-turerangeindicatedand thatforsakeofclarity onlythephase boundaries

areshown.

s

40

30

20

0 0.2 0.4 0.6 08 I0

XDPPC

in

DMPC

FIGURE 8 Phasediagramoffullyhydrated DMPC/DPPC multilamel-lar mixtures. Phase transition temperatures were obtained based on wide-angle x-ray diffraction data recorded using the temperature gra-dient method asdescribed in the text. The low temperature phase is

designated "gel"sinceanindependentdeterminationof its lattice type andsymmetrywas notmade in theseexperiments.Phaseboundariesare

is reflected in Fig. 7 by the lower onset temperatures using low-angle data and the higher completion temperatures usingwide-angle information.

As an independent check on the validity and internal consistency of the phase diagram obtained using the temperature gradient method, we verify the following prediction. The structural parameters observed for theLa and

Lg.1

phases in the coexistence region of Fig.7should be thoseof the corresponding solidus and fluidus compositions at agiven temperature and overall lipid composition. The data in Fig. 7 indicate a fluid and gel phase composition

(XDPPE)

of 0.4 and

0.7,

respectively,

at550C andanoverall

lipid composition of 0.6. Referring to Fig. 5 we see that at 550C samplesof composition 0.4 and 0.7 have interlamel-lard-spacing values of62.5 and 70A, respectively. These are in excellent agreement with the structural parameters of 63 and 70 A observed in the coexistence region as reported in Fig. 3.

Atthis juncture the advantages and disadvantages of the temperature gradient method and its use in temperature-composition lipid phase diagram construction will be con-sidered. To begin with, the method is intuitively simple (Fig. 1): samples contained in long capillaries arranged around the perimeter of a rod upon which a temperature gradient has been imposed represent isopleths (lines of constant overall composition) in the phase diagram. The time-resolved x-ray diffraction method facilitates rapid phase identification and quantitation in temperature-composition space.

The fact that x-ray diffraction is used to provide phase information offers many advantages. Itgives positive phase identification both insingle and multiple phase regions and allowsus tolocatephase boundaries. Fromthe diffraction dataitself structural parameters of the various phases can be obtainedalong eachisopleth continuouslyas afunction of temperature. The continuous nature of the method is also important because it means that phase fields or coexistence regions that exist over narrow temperature regions will be readily detected. Because the method is quantitative, the relative amounts of phases in phase coexistenceregionscanbedetermined (see Caffrey, 1985). This can be used togreatadvantage inverifying interpo-latedphase boundaries in the phase diagram.

The use of diffraction for phase identification and quantitation also means thatartifacts, which might arise with other methodsduetopreferential sample orientation, especially in one-phase regions, are avoided. We empha-size, however, that this relies on the use of a two-dimensionaldetector. The diffraction method also obviates the need for additives or molecular labels, both of which are potentially perturbing. An added convenience of the present system is that data is recordedon videotape. The data can, therefore, be carefully reviewed at leisure in replay mode at selected speeds and the appropriate seg-mentsidentified for subsequent image processing.

While x-rays at theintensity available through synchro-tron radiation can be damaging (Caffrey, 1984), the temperature gradient method requires that a particular portionof the sample is in the beam for a very short period of time. Thus, the method is nondestructive and the sample canberecovered for reuse. This offsets oneof the disadvan-tagesof the method, namely the need for largeamountsof material. For example, to prepare a fully hydrated DPPC sample 1 mm indiameter and4cm longrequires 30-50 mg lipid.

The main advantage of the method is data acquisition speed. No longeris it necessary to equilibrate samples at each successive temperaturebefore making the diffraction measurements. With samples of different compositions positioned lengthwise around the perimeter of the cylinder in acolt revolver fashion (Fig. 2) all that is required is to allow thesamples to reacha steady-stateon thegradient and to makethe diffraction measurements. This takes on the order of 30-60s/sample. In principle, the data for a complete phase diagram with 15-20 isopleths could be collected by this method in 1 hof beam time. Theability to collectdataquickly alsomeansthatsystematic and nonsys-tematic errors duetosamplesource and preparation, and to data acquisition and analysis procedures are mini-mized.

Finally, the temperature gradient method offers the advantage of scale expansionorcontraction. Forexample, if the intention is to obtain information on the general phase behavior of a system over a wide temperature range, the temperaturegradientcan beso setup. This should be extremely useful in survey type work.If,onthe otherhand, the intent is to investigate a particularly narrow phase coexistenceor transition region, thetemperaturegradient canbe veryconvenientlyexpandedinthe regionof interest for detailed examination. Inthiscontext we notethat it is not necessary to limit oneself to linear temperature gra-dients. Gradients of different profiles are availableby an appropriate choice ofgradientrodshape(Holman, 1976).

On thenegative side, the temperaturegradientmethod isdependentonhavingaccess toitemsofhigh technology. Theseincludea monochromatic x-ray beamofhigh inten-sity of the type that is availableat asynchrotron source,a two-dimensional x-ray imaging device, video equipment and,forquantitativedataanalysis,image processing capa-bilities. Of course, detection, recording, and analysis sys-tems other than the ones described above are possibleand in certain instances may beeven moredesirable.Thepoint is that acomplete system in oneformoranother shouldbe available at each of the synchrotron radiation facilities around the world (Winick, 1980).

alongthe lengthof the capillary. While not a big problem, itdoes lead to gradual changes in diffracted intensity from oneend of the sample to the other.

A rate-limiting step in this procedure is alignment of capillaries, particularly when operating with the colt revolver arrangement shown in Fig. 1. The only way around this is to have available an arsenal ofgradientrods mounted on goniometerheadsuponwhich capillaries have been aligned ahead of time. This will ensure the most efficient use of beam time.

No insulation is provided in the present experimental arrangement,andatemperaturegradientexiststransverse to the long-axis of the capillary (Fig.2). This arises because onlyonesideof the capillary contacts thegradient rod while the other side is exposed to air at ambient temperatures. The netresultis toslightly broaden and to shift transition temperatures since the x-ray beam now samplesadiagonal rather thanaverticalphaseboundary. Enclosing the apparatus in avacuumchamber will elimi-natethis problem. We note,however, that in the tempera-turerangeexamined in the present setofexperimentsthis effect was minimal in terms of measured transition tem-peratures.

Finally, the issue that the temperaturegradientmethod is asteady-state rather than an equilibrium technique must be addressed, particularly since the intent is to use it in constructing phase diagrams that are for the most part considered toreflecta true equilibrium and tofollow the Gibbs phase rule. While it is true that the method is steady-state, the empirical observation is that it works. Phase diagramsobtained by this method are in excellent agreement with those obtained by other more standard "equilibrium" methods. At least in the cases examined, therefore, the steady-state issue does not appear to be a problem.However,it is instructive to examine the situation that prevails, for example, in a two-phase coexistence region upon which a temperaturegradient is imposed. To thisend,letusconsider the phasediagramshown inFig. 1. Wenotethat in the phase coexistence region a concentra-tiongradientof thetwocomponentlipid species (A and B) existsalong thelengthof thecapillary.Forthemoment we will address the situation in the liquid-crystalline fraction since, by comparison,moleculardiffusion in thegel phase isextremely slow (Alecioetal., 1982).ReferringtoFig. 1, we seethattheliquid-crystalline phase is0.7Aand 0.3B at positionainasample whose overallcomposition is equimo-lar in A and B. However, at the higher temperature represented by point b in the capillary, the composition is 0.6A and 0.4B.Thus, for lipidAaconcentration gradient existsfromb to awhereas forlipidBthegradientis in the opposite direction. By extension a concentration gradient exists for bothlipids atallpoints in the phase coexistence region, which should drivelipid diffusion.

Todetermine if this effect brought about any noticeable change in thephase diagram measurements were carried

out on an equimolar mixture of DPPC and DMPC incu-bated on thegradient rod for 15 min and for 2 h. In both cases the onset and completion transition temperatures were the same to withinexperimental error and agreed well with those obtained by DSC (Chapman et al., 1974). It appears, therefore, that lipid diffusion is inconsequential on the time-scale of these experiments. Perhaps it is because lipids are confined to individual multilamellar vesicles between which lipid exchange is slow (Schroit and Madsen, 1983; Tamura etal., 1986) that accounts, at least in part, for this result. Diffusion is more likely to be a problem in systems that exhibit phase coexistence, one phaseof which is an isotropic fluid.

CONCLUSION

A new "steady-state" method isdescribed that combines time-resolved x-ray diffraction and a temperaturegradient for temperature-composition lipid phase diagram construc-tion atunprecedented rates. Themethod has been used to constructphasediagramsfor fullyhydrated DPPC/DPPE and for DPPC/DMPC multilamellar mixtures, which agree with and extend data obtainedbyavarietyofother physical techniques. Advantages of the new method include rapid data acquisition rates and sample through-put, positive phase identification and quantitation, and structural parameter determination continuously with temperature in single and multiple phase regions. The method is also inexpensive,nonperturbing, nondestructive, insensitive to orientation effects, and offers the ability to changethetemperaturegradientrangeandprofileatwill. Further, thetwo-dimensional data recorded on videotape can be examined at leisure in replay mode. The method does, however, require access to a high intensity, mono-chromatic x-ray source and to suitable detection, record-ing, and analysis devices, relatively large amounts of sample,and carefulsamplealignment. It would be greatly improved by the provision of an evacuated sample chamber andby a guaranteeof sample homogeneity.

The authorsthankB. W. Batterman(National Science Foundationgrant

DMR81-12822),D. H.Bilderback, G.W.Feigenson (National Institutes of Healthgrant HL-18255), and J. K. Moffat(National Institutes of HealthgrantsRR-01646andGM29044) for their invaluable help and

support. Further thanks go to W. W. Webb formakingavailable the

Grinnell image processing systemand to EllenPatterson forcarefully typing the manuscript.

This work incorporates theundergraduate seniorthesisofF.S.Hingand

wassupported in part by a Grant-in-Aid of Research from Sigma Xi, the

ScientificResearchSociety, and TheNationalInstitutes ofHealth,grant numberDK36849,to M.Caffrey.

Receivedforpublication 12May 1986and infinalform4September

1986.

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