Pesticide Science Pestic Sci 55 :147–153 (1999)

Effects of the herbicide prometryn on thephysicochemical properties of artificial modelmembranesA Bertoluzza, S Bonora, G Fini* and MA MorelliCentro di Studio s ulla Spettros copia Raman,Department of Biochemis try , Univers ity of Bologna, via Belmeloro 8 /2, I -40126Interfacolta

Bologna, Italy

Abstract : Several derivatives of 1,3,5-triazine (eg atrazine, prometryn) are eþ ective herbicides and

have been extensively used in agriculture. A study of prometryn (Prom)-phospholipid (PL), with dif-

ferent charges, was carried out by means of calorimetric and spectroscopic techniques. The Prom-

dipalmitoylphosphatidylcholine (DPPC) and Prom-dipalmitoylphosphatidic acid (DPPA) water

suspensions systems at pH values ranging from 5 to 9 were investigated. The results show that Prom

does not signiücantly perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide

localizes near the glycerol backbone of the lipid, perturbing the environment of the carbonyls of the

two ester groups. At pH 5 the vibrational band attributable to N–H stretch of Prom disappears thus

suggesting that the absorption into the bilayer modiües the physicochemical properties of the her-

bicide. The two PL considered behave similarly and the eþ ect of the charge is not noticeable. The

strength of the Prom-PL interaction decreases with decreasing pH.

1999 Society of Chemical Industry(

Keywords: phospholipid ; phase transition; herbicides ; triazine; prometryn

1 INTRODUCTION

Several derivatives of 1,3,5-triazine are eþective her-bicides and are extensively used to control weedsselectively in crops, nurseries and orchards, and fornon-selective weed control on industrial sites, rights-on-way and irrigation ditches. The S-triazine com-pounds exert their herbicidal properties byinterfering with the photosynthesis process in plants ;however the details of their mode of action are notfully understood.1 They seem to inhibit basal elec-tron transport, methylamine-uncoupled electrontransport and noncyclic electron transport, withwater as the electron donor and ferricyanide orNADP` as the electron acceptor. These herbicideshave also been supposed to inhibit photosyntheticelectron transport between the primary PS IIelectron-accepting quinone and the two-electrongate, a plastoquinone termed B.2 Studies have indi-cated that the molecular basis for the resistance ofsome plants and algae to the triazine derivativescould be due to a change in the DNA coding for oneof the proteins found in the PS II complex.3h5 Allthese ündings have prompted us to suppose that theinteraction between triazine derivatives and mem-brane lipids could play an important role in the her-bicidal eþects, mainly by modifying the structure ofthe lipoprotein complexes.

Prometryn (N2, N4-di-isopropyl-6-methylthio-1,3,5-triazine-2,4-diamine), a member of the triazinefamily, is a widely used herbicide and is similar toatrazine in biological properties. However, unlikeatrazine, prometryn should not give the hydroxy(bio-inactive) form as a consequence of the proto-nation of amino groups ; indeed the surface aminogroup of atrazine is protonated when exposed toatmospheric moisture;6 the protonation of the aminogroup destabilizes the chloro-atrazine form becausethe electron density in the vicinity of the protonatedamino-group decreases. This renders the CwClbond less stable, and Cl can be easily substituted byOH, thus forming protonated hydroxy-atrazine. Thisfact could modify the mechanism of prometrynaction compared to that of atrazine.

Our previous studies on the interaction betweenbipyridilium derivatives (paraquat, diquat) and phos-pholipids supported the idea that herbicides can havebiomembranes as the primary target for theiraction;7 in particular, paraquat, a herbicide posi-tively charged at pH 7, is involved in an electrostaticinteraction with lipids, and the interaction is particu-larly intensive with negatively charged phospholipids(eg DPPA).

In this paper we report studies on the interactionof prometryn (Prom; Fig 1) with dipalmitoylphos-

* Corres pondence to : Centro di Studio Interfacolta s ullaG Fini,

Spettros copia Raman, Department of Biochemis try, Univ of

Bologna, via Belmeloro 8/2, I-40126 Bologna, Italy

Contract/grant s pons or : MURST(Received 28 July 1997; revis ed vers ion received 23 February

1998; accepted 29 September 1998)

( 1999 Society of Chemical Industry. Pestic Sci 0031–613X/99/$17.50 147

A Bertoluzza et al

Figure 1. Structure of prometryn (Prom)

phatidylcholine (DPPC) and dipalmitoylphosphati-dic acid (DPPA) liposomes for a better evaluation ofthe mode of action of this herbicide.

2 EXPERIMENTAL

2.1 Preparation of liposomes

Phospholipids (PL) were obtained from SigmaChemical and prometryn from Riedel de Haen(Pestanal). All chemicals were used without furtherpuriücation.

Multilamellar liposomes were prepared by mixingchloroform solutions of Prom and PL to reach anappropriate PL/Prom, ratio. The solvent was evapo-rated under a stream of nitrogen and then undervacuum for 4h. The DPPC/Prom mixtures werehydrated by adding the buþer solutions. The buþerswere: citric acid/sodium citrate (pH 5), sodium dihy-drogen phosphate/disodium hydrogen phosphate(pH 7) and boric acid/borax (pH 9). The buþeringchemicals were dissolved in saline solution (sodiumchlorides 9g litre~1) and total amount of buþeringcomponents was about 10~3 M. At these low concen-trations, the IR spectra of the buþers did not appearon the IR spectra of DPPC. DPPA and the DPPA/Prom mixtures were hydrated by adding the appro-priate amount of sodium hydroxide in saline up tothe chosen pH value and then saline solution to givethe same volume in all the samples ; the total ionicstrength of the suspensions was kept constant foreach pH value.

2.2 Differential scanning calorimetry

Diþerential scanning calorimetry (DSC) measuresthe changes in heat capacity of a system with changein temperature. Any change in state in the system isindicated by a sharp peak whose characteristics givean insight to the nature of the change. DSC has beenwidely used to examine phase changes occurring inlipids,8 and is a valuable tool for studying their inter-action with other molecules. The parameters usuallymeasured are the temperature of the transition,Tm ,

the width of the peak at half-height, and *H,W1@2 ,the enthalpy change during the transition, deter-mined from the area under the peak.

The lipid/water suspensions were heated in awater bath at about 10¡C above the phospholipidtransition temperature for 2h.

Exactly weighed amounts (10mg) of each lipidsuspension were encapsulated in ýat-bottomed alu-minium pans of 40ll with crimped on lids.

Diþerential scanning calorimetric measurementswere made in air with a Mettler DSC 20 calorimeterin the 25–95¡C temperature range. Heating runswere performed at 2¡Cmin~1. Before each heatingrun the samples were allowed to stand at 25¡C for30min, and heating runs were repeated three timesto ensure reproducibility of the thermal parametersof the main transition. The quantity of PL used foreach calorimetric measurement has been determinedcolorimetrically as reported elsewhere7 and in the lit-erature cited therein.

*H of fusion was determined by integration of theareas under the melting DSC endotherms followingcalibration with indium. The errors in enthalpy wereless than 5%.

Caprylic acid was used to calibrate the tem-perature in the range studied.

2.3 Spectroscopic measurements

Vibrational Raman spectra were recorded with aBruker IFS 66 spectrometer equipped with aFRA-106 Raman module and a Ge diode detector.The excitation source was a Nd3`-YAG laser(1064nm) in the backscattering (180¡) conüguration.The focused laser beam diameter was B100lm, thespectral resolution 4cm~1, the encoding interval 1point cm~1 and the apodization function BlackmanHarris, 4-term. The laser power was 250mW on thesample.

Infrared spectra were recorded by a JASCO FT/IR-300E. The spectra were obtained with the ATRtechnique by using a ZnSe plate (this techniqueallows aqueous solutions to be studied easily). Inorder to obtain good quality spectra, 1000 scans foreach spectrum were accumulated and computer aver-aged. The errors in wavenumber were about0.4cm~1.

The spectra of the buþers and of water were com-puter subtracted to obtain the liquid or the lipid-Prom complex spectra.

UV measurements were carried out by means of aJASCO 7850 UV-vis spectrophotometer.

All spectra were measured at room temperature.

3 RESULTS AND DISCUSSION

3.1 Dipalmitoylphosphatidylcholine (DPPC)

DPPC is one of the most common components ofbiological membranes and can be assumed as a phos-pholipid model for the uncharged part of a biologicalmembrane. Multilamellar liposomes obtained byDPPC water suspensions are very extensively used asa good model for biological membranes.

The calorimetric data of DPPC and the DPPC–Prom systems (Prom/DPPC molar ratio ranging

148 Pestic Sci 55 :147–153 (1999)

Eþ ects of prometryn on properties of biomembranes

Table 1. Calorimetric data of DPPC/Prom s ys tem

pH Tm(¡C) W

1@2(¡C) *H (kJmolÉ1) Tpt r(¡C) Molar ratio (Prom /DPPC)

DPPC 5 41.3 0.7 34.1 36.0 –

DPPC 7 41.4 0.5 34.1 33.9 –

DPPC 9 41.3 0.7 34.2 34.8 –

DPPC]Prom (0.5%wt) 7 41.1 0.5 34.2 32.1 0.015

DPPC]Prom (1%wt) 7 41.0 0.9 34.4 31.6 0.030

DPPC]Prom (2%wt) 7 40.9 1.0 34.2 31.2 0.060

DPPC]Prom (5%wt) 7 40.7 1.1 34.4 31.2 0.150

DPPC]Prom (10%wt) 7 40.0 1.2 34.5 30.5 0.300

DPPC]Prom (20%wt) 5 40.7 1.2 33.2 – 0.600

DPPC]Prom (20%wt) 7 40.0 1.2 34.6 – 0.600

DPPC]Prom (20%wt) 9 39.5 1.2 34.8 – 0.600

DPPC]Prom (40%wt) 7 40.0 1.2 34.6 – 1.200

from 0.015 to 1.2) are given in Table 1. For DPPCthe temperature of the main transition is 41.4¡C(Tm)and the associated enthalpy change, *H, 34.1kJmole~1.

In the presence of increasing amounts of Prom, aslight decrease in and a more noticeable loweringTmin the temperature of the pre-transition were(Tptr)observed; moreover, the pre-transition peak was nolonger present when the concentration of Promreached 20% w/w. These results are in agreementwith the idea that, in most cases, the eþect of theaddition of chemicals to lipid bilayers is more pro-nounced on the pre-transition than on the main tran-sition.8

At pH 7 the main transition peak was fairly sym-metrical and its shape did not change as a conse-quence of the Prom addition, thus indicating that theaffinity of Prom toward the hydrophobic moiety ofthe liposomes in the gel and the liquid crystallinephase is the same.8 Moreover, the addition of Prom(20% w/w) to neat DPPC caused an increase in themain transition half-width from 0.5¡C to(W1@2)1.2¡C. Because of the approximately inverse relation-ship between the number of PL molecules per coo-perative unit and the observed broadeningW1@2 ,9indicates a noticeable lowering of the transition coo-perativity of DPPC due to its interaction with Prom.

Changes of pH in the pure DPPC water suspen-sion did not change either the transition temperatureor the proüle of the endothermic transition, as wouldbe expected for uncharged molecules of DPPC in the5–9 pH range. On the other hand, pH changes in thesame range aþected the DPPC–Prom interaction(Table 1); indeed, the temperature of the main endo-thermic transition increased as the pH was loweredto acid values.

Since the pKa value for Prom is 4.110 at pH 5,only a small portion of Prom molecules should beprotonated (about 1/10). This is also supported bythe infrared spectra of Prom, in which the absorptionbands due to the protonated form do not appear, andthe spectrum at pH 5 is almost the same as at pH 7and 9.

Consequently, the DPPC–Prom interaction shouldnot depend on pH in the range under consideration.However, the calorimetric results indicate that thebehaviour of Prom at pH 5 is quite diþerent fromthat at pH 7 and 9.

The thermograms observed after the addition ofProm at pH 9 were slightly skewed towards highertemperatures, suggesting that Prom is distributedpreferentially into the gel regions in the membrane.8

The lowering of the phase transition temperature(with an increase in pH) without changes in the tran-sition enthalpies indicates that there is an interactionof the herbicide with the polar head group region ofDPPC and that a partial penetration of Prom into thehydrocarbon region of the liposomes took place.

In addition, inspection of the calorimetric datareveals that, since an amount of Prom greater than20% w/w does not inýuence the PL properties, somesaturation eþect has taken place.

These ündings were conürmed by Raman spec-troscopy. This spectroscopic technique gives twoorder parameters related to the trans/gauche ratio ofthe acyl chains (C–C stretching region) and thelateral packing of the acyl chains (C–H stretchingregion).7 The trans/gauche ratio both in the gel andliquid-crystalline phase did not change as a conse-quence of Prom addition (20% w/w); the lateralpacking decreased by about 5% (from 0.37 to 0.35)in the gel phase and remained constant in the liquid-crystalline phase. These data also conürm that theinteraction between DPPC and Prom is limited tothe polar moiety of the bilayer.

Vibrational spectroscopy gives further, moredetailed insight into the type of interaction betweenthe lipid and the herbicide. The IR and Ramanspectra of DPPC and DPPC–Prom water dispersion(20% w/w) at pH 7 are shown in Figs 2(A) and 3(A).Since the DPPC molecules have two carbonylgroups, a doublet should be observed in the carbonylstretching region; however it has been widelydemonstrated that conformational eþects are negligi-ble and the presence of more than one band is pri-marily due to hydrogen bonding and the polarity in

Pestic Sci 55 :147–153 (1999) 149

A Bertoluzza et al

Figure 2. (A) Infrared s pectra of the CxOs tretching modes of (a) pure DPPC and

DPPC Prom (20%wt) water s us pens ions at(b) pH 9, (c) pH 7 and (d) pH 5, and (B)

their s econd derivative s pectra.

the immediate vicinity of the carbonyl groups.11h13Only one very broad and fairly symmetric bandappeared in both the IR and Raman spectra of neatDPPC at 1734cm~1 and 1738cm~1, respectively.Furthermore, the second derivative spectra showedonly one main component (Figs 2B(a) and 3B(a)).The two carbonyl groups should be equally acces-sible to water and their chemical environments areequivalent. On addition of Prom (20% w/w), theshape of the vCxO stretching band became asym-metric; the maximum wave number of the IR bandremained constant and the Raman wave numberdecreased to 1735cm~1. This behaviour can beexplained by considering that the asymmetric band isan overlap of two main components whose intensitieschange as a consequence of the interaction withProm. These two main components can be distin-guished by means of the second derivative spectra(Figs 2(B) and 3(B)). The presence of only one maincomponent in the IR and Raman spectra of neatDPPC and of two main components in the DPPC–Prom spectra indicates that the environments of thetwo CxO groups of neat DPPC are approximatelyequivalent, but in the presence of Prom this is nolonger the case. This indicates that Prom does notpenetrate deeply into the bilayer and is able to

modify only the chemical environment of the sn-2carbonyl group closer to the bilayer interface.14

In the N–H stretching region of the IR andRaman spectra of Prom (pH 7) two bands appearedat 3234 and 3091cm~1 (Figs 4(a) and 5(a)). Thesespectral features are not aþected by pH changes ;indeed, the same spectra are obtained at pH 5 andpH 9. This splitting cannot be attributed to the pres-ence of two N–H groups. Indeed, as a consequenceof deuteration, only the ürst component was shiftedto lower wavenumbers : the spectrum of the deuter-ated Prom exhibited a band at 2373cm~1, whichagrees well with the calculated value of 2362cm~1.The low wave number component can be attributedto anharmonic resonances. Possible candidates forthe summation tones participating mainly in the lowfrequency component of the doublet are1506] 1588\ 3094cm~1, which are present atabout the same wavenumbers in the spectrum of thedeuterated Prom.15

The relatively low value of the N–H stretchingwavenumber indicates that neat Prom is hydrogenbonded as a consequence of the presence of the N–Hgroups in the molecule. Indeed, vN–H increases to3440cm~1 in diluted carbon tetrachloride solution,where hydrogen bonds are not present.15 As a result

Figure 3. (A) Raman s pectra of the CxOs tretching modes of (a) pure DPPC and

(b) DPPC/Prom (20%wt) water s us pens ionsat pH 7 and (B) their s econd derivative s pectra.

150 Pestic Sci 55 :147–153 (1999)

Eþ ects of prometryn on properties of biomembranes

Figure 4. Infrared s pectra of the N–H s tretching mode of (a) pure

Prom in water s us pens ion at pH 7 and Prom in DPPC/Prom(20%wt) water s us pens ions at (b) pH 7 and (c) pH 5.

Figure 5. Raman s pectra of the N–H s tretching mode of (a) pure

Prom in a water s us pens ion at pH 7 and Prom in DPPC/Prom(20%wt) water s us pens ions at (b) pH 7 and (c) pH 5.

of the presence of DPPC at pH 7, the vN–H did notchange signiücantly compared to that of neat Prom(Figs 4(b) and 5(b)). This fact suggest that thestrength of hydrogen bonds in neat Prom and in theDPPC–Prom system is about the same.

The wavenumber of vN–H did not change byincreasing pH to 9 either in the spectrum of neatProm or in that of Prom interacting with DPPC.

In the IR and Raman spectra of the DPPC–Promsystem at pH 5, the band attributable to vN–Hstretching was no longer present (Figs 4(c) and 5(c));a broad absorption, which modiües the base line ofthe IR spectrum, was observed around 3000cm~1.This absorption is typical of protonated aminogroups.16 This fact suggested that the DPPC–Prominteractions change when pH is lowered to acidicvalues. At these pH values, the protonation of Promseems to take place. This result is in agreement withthe calorimetric data and caused us to suppose thatthe basicity of Prom increases upon interaction withDPPC.

3.2 Dipalmitoylphosphatidic acid (DPPA)

In resting cells, phosphatidic acids constitute aminor portion of the total phospholipid pool, withmoderate biological reactivity. However, being nega-tively charged at physiological pH, they are veryimportant in interactions with positively chargedcations, such as polyamines17,18 and some herbicides(paraquat, diquat).7 The functions of phosphatidicacids as intracellular mediators and extracellularmessengers of biological activities19 have also beenreported.

At pH 5 and pH 7 the DPPA molecules bear onenegative charge, due to the partial ionization of thephosphate group.20 The complete ionization of thephosphate group occurs at pH 12.21 Since the totallipid charge depends on pH, the temperature of themelting transition and the associated value of *Halso depend on pH (Table 2). The transition tem-perature of DPPA decreases with increase in pHaccording to the literature data.20h22 As a conse-quence of the Prom addition, a slight decrease of Tmwas observed: this decrease was 1.7¡C at pH 5 and3.8¡C at pH 9. A qualitatively similar behaviour wasobserved in the DPPC–Prom systems.

The of the transition remained unchangedW1@2suggesting that the addition of Prom is not able to

pH Tm(¡C) W

1@2(¡C) *H (kJmolÉ1)

DPPA 5 68.1 3.7 26.4

DPPA 7 64.9 3.6 23.5

DPPA 9 57.5 4.1 21.0

DPPA]Prom (20%wt) 5 66.4 3.6 25.5

DPPA]Prom (20%wt) 7 62.1 3.7 23.7

DPPA]Prom (20%wt) 9 53.7 4.2 22.3Table 2. Calorimetric data of

DPPA/Prom s ys tem

Pestic Sci 55 :147–153 (1999) 151

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change markedly the cooperativity of the transitionof DPPA.

The IR and the IR second derivative spectra ofDPPA and DPPA–Prom at pH 7 in the vCxOstretching region are illustrated in Fig 6. The bandattributable to the vCxO stretching mode of theDPPA ester groups is very broad. A slight shifttowards low wavenumbers was observed in the pres-ence of Prom. It seems that Prom aþects both car-bonyl groups equally, thus indicating that thepenetration of Prom into the bilayer can reach bothcarbonyl groups of DPPA. Indeed, the second deriv-ative spectra showed only one main component in allcases (Fig 6B). This diþerent behaviour could be dueboth to the expansion of the lipid lattice due to nega-tive charges, which facilitates the insertion of theherbicide into the bilayer and to the diþerent struc-ture of the glycerol backbone in the two lipids.23

The spectra of Prom in the vN–H stretching rangedoes not change on the addition of DPPA in the 7–9pH range; the band attributable to lN–H disappearsin the presence of DPPA at pH 5.

The vibrational results agree well with those ofDPPC and it appears that the two lipids interact withProm by an analogous mechanism.

4 CONCLUSIONS

The calorimetric and spectroscopic results show thatProm does not perturb the hydrophobic core of thephospholipid (PL) bilayer. As regards DPPC, only aslight decrease of the temperature of the meltingprocess was observed. The *H associated with thetransition remained constant ; the order parametersof acyl chains calculated by means of Raman spec-troscopy showed only a very small decrease, whichwas not particularly signiücant. This behaviour is inagreement with the results reported by other authorson DPPC-atrazine systems.24,25

In the vCxO stretching region of the DPPCspectra, a broad band appeared. Its wavenumberdecreased in the Raman spectrum after Prom addi-tion. By means of the second derivative spectra,

which allow spectral diþerences arising from smallvariations in the structure at the sub-molecular levelto be detected and assessed, two main componentscan be identiüed. The presence of two main com-ponents indicates that the environments of the twoC\ O groups of DPPC are no longer equivalent ;Prom penetrates into the bilayer only until it reachesthe more accessible carbonyl group, while the moreinternal group interacts mainly with the water mol-ecules, as observed in the absence of Prom.

The partition coefficient of Prom (octan-1-ol/water) as deduced by UV measurements depends onthe pH of the aqueous solution. At the three con-sidered pH values, comparable results were obtainedfor pH 7 and 9 (2] 103). At pH 5 a signiücantlylower value has been measured (5] 102); however inthis case as well we can suppose that the herbicidecould interact with biomembranes. Our results indi-cate that this interaction is mainly related to thesuperücial part of the bilayer, with a stronger inýu-ence on the CxO ester group nearest to the liposomesurface (sn-2).

Calorimetric data indicate that the thermal behav-iour of neat DPPC liposomes is not aþected by thepH changes ; however, a decrease in pH alters thethermal behaviour of the DPPC–Prom systems. AtpH 7 and pH 9 Prom is uncharged and is much moreeþective in lowering the melting temperature ofDPPC than at pH 5 when the modiüed form ispresent, as suggested by the vibrational spectra inthe N–H stretching region.

Since the pKa value of Prom indicates that only asmall percentage should be protonated in aqueoussolution at pH 5, the spectroscopic behaviour oflN–H can be explained only by supposing that theinteraction of Prom with the glycerol backbone ofPL strongly increases the basicity of the N–Hgroups.

DPPA, was studied since its negatively chargedPL were found to strongly interact with aminogroups of polyamines17,18 and of some herbicides.7However, Prom interacts similarly with DPPC andDPPA, and the negative charge on PL seems have anegligible eþect.

Figure 6. (A) Infrared s pectra of the CxOs tretching modes of (a) pure DPPA and

DPPA/Prom (20%wt) water s us pens ions at(b) pH 9, (c) pH 7 and (d) pH 5 and (B) their

s econd derivative s pectra.

152 Pestic Sci 55 :147–153 (1999)

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Moreover, it is interesting to note that some herbi-cides interact mainly with the negatively chargedPL ;7 on the other hand, atrazine24,25 and Prom alsointeract with uncharged PL. The interaction betweenProm and PL is limited mainly to the superücial partof the bilayer. Prom is able to modify the environ-ment of only one ester group in DPPC.

The PL–Prom interaction seems to increase thebasicity of the N–H groups of the triazinic herbicide.Analogous eþects on the acid-base properties ofProm could take place in the soil by adsorption of theherbicide molecules on clay particles, as has beenreported for atrazine.6

As a consequence of the changes on lipidic mem-brane caused by the interaction of Prom with PL,basic mechanisms operating at the membrane levelcould be aþected, for example, the permeability ofliposomes to non-electrolytes and the electrolyte dif-fusion. These interactions could also perturb normallipid–protein interactions in the modulation ofenzyme activity.

ACKNOWLEDGEMENT

This work was supported by grants from MURST60% and 40% to GF.

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