Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

Chemistry and Physics of Lipids, 22 (1978) 39-50 © Elsevier/North-Holland Scientific Publishers Ltd.

HORMONE (3-1NDOLEACETIC ACID)-PHOSPHOLIPID INTERACTION: IH, 13C AND alp NUCLEAR MAGNETIC RESONANCE STUDIES A. MARKER, L.G. PALEG and T. McL. STOPSWOOD* Department of Plant Physiology, WaiteAgricultural Research Institute and *Department of Organit Chemistry, The University of Adelaide, Adelaide, South Australia 5000 (Australia} Received August 22nd, 1977

accepted November 16th, 1977

The interaction between the plant hormone, 34ndoleacetic acid (IAA), and some phospholipids in CDCL3 has been studied by 1H, laC and 31p nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidyleholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (Kd). In all cases, Kd values were approx. 10-2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of IAA.

I. Introduction As part o f the continuing studies on plant hormones [ 1 - 4 ] , we have undertaken a programme o f biophysical studies o f interactions o f plant hormones and membrane components using nuclear magnetic resonance (NMR) spectroscopy. Earlier work with IH NMR provided evidence o f interactions between purified plant phosphatidylcholines and gibberellic acid [3,4] and 3-indoleacetic acid (IAA) [4] in deuterochloroform (CDCla) solutions. We have now extended the work to a detailed study o f interactions in CDCIa solutions between IAA and phospholipids, using ~3C and 31p NMR, as well as ~H NMR.

II. Experimental A. Materials Synthetic 1,2-dipalrnitoyl-3-sn.phosphatidylcholine (DPPC) was obtained from Calbiochem (Lot 300319). Plant source (soy bean) phosphatidylcholine (PC(P.L.)) and phosphatidylethanolamine (PE(P.L.) were obtained from P.L. Biochemicals (Lots 471-1 and 471-4, respectively). These lipids were used without further purl39

40

A. Marker et aL, NMR'studies of hormone (IAA J-phospholipid interactions

Table 1 Fatty acid composition of phospholipids Phospholipid

DPPC PC(P.L.) PE(P.L.) PE (Sigma)

% Fatty acid 16:0

18:0

100 15.3 23.2 23.5

. 4.9 3.1 2.6

18:1 .

. 9.3 6.3 10.1

18:2

18:3

63.8 60.5 58.7

6.7 6.9 5.1

.

fication. A further lot o f plant source phosphatidylethanolamine (PE(Sigrna)) was isolated from Sigma crude soybean lecithin (Lot 12C3010) by alumina and silicic acid column chromatography. All lipids gave single spots on silica gel H thin layer chromatograms using chloroform-methanol-acetic acid-water (50 : 25 : 7 : 3) as the solvent system. The fatty acid composition of the phospholipids, determined by GLC (15% diethylene glycol succinate (DEGS) on Chromasorb W packing) of the methylated fatty acids after hydrolysis and methylation by treatment with 5% H2SO4 in anhydrous methanol at 70°C for three h under N2 (A. Sinclair, personal communication), is given in table I. IAA (BDH Laboratory Reagent) was recrystallised from 1,2-dichloroethane [5], and the CDCI3 was obtained from C.E.A. (France). Sample preparation for NMR measurements usually involved preparation of a solution of known molality of lipid in CDCla, known amounts of which were then added to NMR tubes containing previously determined amounts of IAA. In some cases, lipid and CDCla were separately weighed into NMR tubes, followed by successive additions of known amounts of IAA: spectra were recorded after each addition. B. Measurement o f NMR Spectra

~H NMR spectra were obtained on a Varian T-60 spectrometer at 60 MHz, with a probe temperature of approx. 40°C, operating in unlocked mode with spectral widths of 250 or 500 Hz and sweep times of 50 s. Chemical shift reproducibility was within -+ 0.03 ppm relative to the internal reference (either residual CHCla or trimethylphosphate (TMP)). ~aC and 3ap NMR spectra were obtained with a Bruker HX-90E spectrometer at frequencies of 22.625 and 36.430 MHz, respectively, in the pulsed Fourier transform mode with broad-band proton decoupling, at a probe temperature of approx. 30°C. For 13C, the spectral width of 5 kHz was accumulated in 8K memory addresses resulting in a chemical shift uncertainty of -+ 0.05 ppm relative to the internal reference (center line of CDCI3 triplet). The spectral width of the 31p spectra was 1.2 kHz and accumulation into 2K memory

A. Marker et al., NMR studies of hormone (IAA)-phospholipid interactions

41

addresses resulted in a chemical shift uncertainty of + 0.03 ppm relative to the internal reference (TMP). TMP was found to be a suitable internal reference for a~p NMR of phospholipids. There was no evidence of interaction between TMP and the phospholipids or IAA. TMP is a relatively chemically stable liquid which is soluble in a variety of organic solvents and in water. The a~p resonance position of TMP, 2.4 ppm upfield from the usual external 85% HaPO4 reference [6], is conveniently near those of phospholipids, and it would not be expected to interfere with any phospholipid spectra. C Calculations

The method of Nicholson and Spotswood [7] and Sykes [8] was used to calculate dissociation constants (Kd) from the experimentally observed variation of phospholipid resonance positions with IAA concentration for 1 : 1 stoichiometries. This procedure was modified for the K d calculations for other stoichiometries (E.H. Williams and T.M. Spotswood, personal communication). For 1 : 1 stoichiometry, under conditions of rapid exchange of IAA between free and lipid bound (HL) states, the observed chemical shift (8obs) of the monitored lipid resonance is related to the change in chemical shift on complex formation (A) and the chemical shift of the free species (~free) by ~obs = ([HL]/Lo] )A + ~free

where [HL] and [Lo] are the concentration of the hormone-lipid complex at equilib. rium, and the total concentration of lipid, respectively.

ilI. Results A. ~HNMR Measurements

Three phospholipid/IAA systems were studied by 1H NMR. The addition of IAA caused upfield changes in the chemical shift of the -~(CH3)3 group of PC(P.L.) of --0.35 ppm and of the -~ H3 group of PE(P.L.) and PE(Sigma) of--0.50 and -0.48 p~_m, respectively (table 2). The magnitude of the change in chemical shift of the -N(CH3)3 group was very similar to that found previously [4], and also to that found by Bray et al. [9] for the association of indole and 3-methylindole with egg lecithin (for corresponding stoichiometries). The upfield shifts of both -/~(CH3)3 and -~H 3 resonances are most likely to be caused by interaction with an adjacent aromatic ring of IAA, and possible ways in which this effect is brought about are discussed below. B. IaC NMR Measurements

The changes in -I(/(CH3)Bgroup 13C resonances of both DPPC and PC(P.L.) induced

42

A. Marker et al., NMR studies o f hormone (1AA)-phospholipid interactions

Table 2 IH Chemical shifts of -N(CH~) 3 and -I~IH~groups of phospholipids as a function of IAA eoncen tration PC(P.L.) (0.030 molal)

PE(P.L.) (0.034 molal)

PE (Sigma) (0.040 molal)

[IAA] (molal)

~H Chemical shift of -I~1(CH3)3a

[IAAI (molal)

1H Chemical shift of -~Hs b

[IAA] (molal)

1H Chemical shift of q~/H~b

0.0000

-0.28

0.0025

-0.32

0.0067 0.0109 0.0151 0.0269 0.0319 0.0369

-0.37 -0.43 -0.46 -0.57 -0.62 -0.63

0.0000 0.0040 0.0069 0.0110 0.0156 0.0271

1.20 1.09 1.00 0.85 0.75 0.70

0.0000 0.0023 0.0045 0.0082 0.0123 0.0149 0.0161 0.0195 0.0200 0.0249 0.0384

1.13 1.10 1.07 1.02 0.97 0.89 0.89 0,86 0.82 0.73 0.65

a In ppm relative to upfield peak of TMP doublet. Negative sign indicates resonance upfield of reference. b In ppm relative to residual CHC13 resonance.

Table 3 ~3C Chemical shifts of -~(CH~)a groups of phospholipids as a function of IAA concentration DPPC (0.087 molal)

PC(P.L.) (0.033 molal)

I IA A I (molal)

~3C Chemical shift of a -N(CII~) s

[ IAA 1 (molal)

~3C Chemical shift of ~ a -N(CHs) s

0.0000 0.0050 0.0070 0.0145 0.0205 0.0420 0.0901

-22.72 -22.75 -22.76 -22.83 -22.88 -23.11 23.40

0.0000 0.0016 0.0043 0.0072 0.0106 0.0180 0.0225 0.0272

-22.55 -22.61 -22.66 -22.72 -22.77 -22.88 -22.93 -22.99

a In ppm relative to central line of CDCI~ triplet. Negative sign indicates resonance upfield of reference.

A. Marker et al., NMR studies o f hormone (1AA)-phospholipid interactions

43

by varying concentrations of 1AA are given in table 3. The 13C chemical shift values of the various carbon atoms of DPPC and PC(P.L.) alone and in the presence of an approximately equimolar concentration of IAA are recorded in table 4. The carbon resonances were assigned on the basis of previous proposals [10-12] which were supported by selective proton decoupling and TI measurements. A later and slightly different assignment [13] introduces uncertainty into the assignment of choline -CH2-O-P and glycerol -CH20-P resonances, but we have adopted the first mentioned assignments in the following discussion. It is clear that the head groups of the lipids are major sites affected, and for DPPC and PC(P.L.), respectively, the following changes occur: the -~(CH3) 3 resonances are shifted 0.7 and 0.5 ppm to higher field; the -~-CH2 - resonances 0.6 and 0.4 ppm to higher field, the choline -CH2-O-P resonances 0.3 and 0.5 ppm to higher field, and smaller upfield shifts are observed for CI and C2 of the glycerol groups (0.1 and 0.2 ppm respectively, for both lipids), whereas the glycerol C3 resonances shift down field 0.4 and 0.3 ppm. In contrast, the resonances of the carbons of the fatty acids show no observable shifts, with the exception of a small downfield shift for both C1 carbonyl carbons of DPPC and one of the carbonyl carbons of PC(P.L.). The large up field 13C shifts in the choline group induced by IAA are paralleled by the large upfield shifts of the proton resonances of the -~(CH3)3 group (table 2), and, for example, with PC(P.L.), the observed 1H and 13C shifts are 0.35 and 0.44 ppm, respectively. We proposed previously [4,14] that the origin of the shift in the proton spectrum lay in the association between the -/~1(CH3)3group and the aromatic ring of IAA. The magnitude of the observed proton shifts implies a specific placement of the plane of the indole nucleus relative to the -~(CH3)3 (or, in the case of the phosphatidylethanolamines, the -I~Ha) group, so that the nitrogen-containing groups are strongly influenced by the diamagnetic ring current of the aromatic ring. Such an explanation could also account for the observed 13C shifts since evidence for ring current effects in 13C NMR has been reported [ 1 5 - 1 8 ] , and since the magnitude o f the effects on both the IH and ~3C shifts is comparable. 13C chemical shifts are also extremely sensitive to changes in local charge density, bound angle deformation and steric compression [19] and though the latter two effects are likely to be minimal, local charge density changes in the phospholipid head group may contribute to the observed shifts. The indole ring system is known to be a strong electron donor in charge-transfer complexes [20,21] and may act as a negative site for ion pairing with -I~(CH3)3 [9], in which case consequent electron redistribution within the choline/ phosphate dipole could contribute to the observed upfield shifts of both ~H and 13C resonances. For instance, in the case of the phosphorylcholine-binding mouse myeloma protein M603, Goetze and Richards [22] suggested that the ~3C upfield shift of 0.7.ppm of the -1~I(CH3)3group is a consequence of reduction of effective charge on the carbon nucleus upon interaction with negatively charged amino acid residues of the protein. However, it should be noted that the -~(CH3)3 group is in close van der Waals contact with the aromatic ring of a tyrosine residue of the protein [23], so that a ring current contribution to the observed shift can not be excluded. Both ring

54£ 66.6 b 59.5 c'd 63.5 b ,d 70.8 e 63.2 173.7,173.3 f 34.5, 34.3 f 25.1 29.8 32.1 22.8 14.2

-N-CH3 -N-CH~chofine -CH2-O-P glycerol -CH:-O~P glycerol -CH-O glycerol -CH2-O palmitate C1 palmitate C2 palmitate C3 palmitate C4~213 palmitate C14 palmitate C 15 palmitate C16

53.8 66.0 b 59.2 b ,d 63.9 b ,d 70.6 e 63.1 173.8,173.5 f 34.5, 34.3 f 25.1 29.9 32.1 22.8 14.2

~aC Chemical shift a (DPPC(0.087 Molal) +IAA (0.090 Molal) -N-CH 3 -N-CH2choline -CH2-O-P glycerol 4SH~-O-P glycerol -CH-O glycerol -CH~-O linoleate C1 linoleate C2 linoleate C3 linoleate C 4 ~ 7 , C15 linoleate C8, C14 linoleate C9, C 13 linoleate CI0, C12 linoleate C 11 linoleate C 16 linoleate C 17 linoleate C 18

Carbon atom

a Chemical shifts in ppm. Converted to TMS scale using 6TMS = 8CDCI~ + 77.2 p'pm. b Resonance broadened, no resolved coupling. c2j13c 3tp~ 5 Hz. d Assignments interchanged in previous communication [14]. e3Jt3c 31p ~ 7 Hz. f Two resolved resonances, non-equivalent carbons. g 3J13c_31P --- 5 Hz.

~3C Chemical shift a DPPC (0.087 molal)

Carbon Atom

~aC Chemical shifts of DPPC and PC(P.L.) and the effects on shifts caused by interaction with IAA

Table 4

54.7 66.6 b 59.7 b 63.4 b 70.8g 63.2 173.7,173.3 f 34.5, 34.3 f 25.1 29.8, 29.4 27.4 130.4,130.1 128.2,128.0 25.8 31.7 22.7 14.2

~aC Chemical shift a PC(P.L.) (0.033 molal)

63.1 173.7,173.4 f 34.5, 34.3 f 25.1 29.8, 29.4 27.4 130.4,130.1 128.2,128.0 25.8 31.7 22.8 14.2

7o.6g

54.2 66.2 e 59.2 b 63.7 b

3C Chemical shift a PC(P.L.) (0.003 molal) +IAA (0.027 molal)

¢5

.Ix -Ix

A. Marker et al., NMR studies o f hormone (1AA).phospholipid interactions

45

current and charge effects should operate in the same direction and could not easily be differentiated. At the highest concentrations of IAA used, the lac IAA resonances were resolved and assigned by comparison with published chemical shifts of similar compounds [24,25]. Due to the low solubility of IAA in CDCI3, a 13C NMR spectrum of ethyl indole-3-acetate was measured to assist with the assignment of IAA. Table 5 contains the laC chemical shifts of IAA and illustrates the effects of DPPC upon the 13C spectrum of IAA. The effect of PC(P.L.) on 13C resonances of 1AA did not differ significantly, even though IAA exerted differential effects on the chemical shifts of the head group nuclei of the two phosphatidylcholines. The chemical shift changes tend to indicate involvement of the whole hormone molecule in complex formation. An interesting observation concerning 13C spectra of IAA was that resonances due to C8 and C9 were not detected in the spectrum of IAA alone in CDCI3, but were clearly resolved in IAA/phospholipid solutions in CDC13. For IAA alone, the observa tion is consistent with low solubility, coupled with long T~ values of these nuclei. For indole, the corresponding C8 and C9 nuclei have TI values of 83 and 85 s, respectively, at 22.6 MHz, whereas TI values of other carbons are in the range 5.5-6.4 s at this frequency [26]. In IAA/phospholipid solutions, the large relative increase in C8 and C9 signal intensities must be due to a reduction in T1. Possible causes of this reduction are a significant increase in molecular correlation times [27]

Table 5 a3CChemical shifts of IAA and effects on shifts caused by interaction with DPPC Carbon Atom

2 3 4 5 6 7 8 9 -CH2C=0

13CChemical shiftsa IAA (saturated solution)

IAA (0.090 molal) + DPPC(0.087 molal)

Change causedby DPPC

123.5 108.1 119.1 122.6 120.2 111.4 b b 31.0 176.0

124.5 108.7 119.3 121.8 119.3 112.2 136.5 127.6 b 174.9

1.0 0.6 0.2 -0.8 -0.9 0.8 -1.1

a Chemical shifts in ppm. Converted to TMS scale using 6TMS = 6CDCla+ 77.2 ppm. b Not resolved.

3.74 3.87 3.90 3.93 4,03 4.16 4.19 4,32 4.45 4.61

0.0000 0.0018 0.0026 0.0042 0.0078 0.0121 0.0146 0.0187 0,0278 0.0.446

a In ppm, relative to TMP.

[IAA] (molal)

31p Chemical shift a

[IAA] (molal)

0.0000 0.0025 0.0067 0.0109 0.0151 0.0269 0.0319

PC(P.L.) (0.030 molal)

DPPC 0.041 molal)

2.95 3.08 3.22 3.28 3.35 3.55 3.59

aap Chemical shift a 0.0000 0.0040 0.0069 0.0110 0.0156 0.0271

[IAA] (molal)

PE(P.L.) (0.034 molal)

31p Chemical shifts of phospholipids as a function of IAA concentration.

Table 6

2.36 2.60 2.73 2,90 3.00 3,20

a~p Chemical shift a 0.0000 0,0023 0.0045 0.0082 0.0123 0.0161 0.0195 0.0200 0.0249 0.0384

[IAA] (molal)

PE(Sigma) (0.040 molal)

2.71 2.75 2.84 2.90 3.00 3.00 3.06 3,06 3.11 3.15

31p Chemical shift a

¢a

O~

A. Marker et al., NMR studies of hormone (IAAJ-phospholipid interactions

47

or relaxation by adjacent -I~(CHa)a protons upon complex formation. Adjacent -I~I(CHa)3 protons are known to contribute to IH-31P dipolar relaxation in phospholipid systems [28,29]. C 31p N M R Measurements In all cases, IAA caused a significant downfield shift o f the a~p resonance o f each o f 4 phospholipids, the magnitude o f which was different for each phospholipid (table 6). The results are consistent with hydrogen bond formation at a phosphate oxygen which has been found to produce downfield shifts o f the alp resonances in phosphatidylcholine and phosphatidylethanolamine extracted from bovine liver [30]. Linewidths in alp NMR spectra o f aqueous dispersions o f phospholipids (unsonicated or sonicated) are strongly dependent upon the physical state o f the phospholipids, and~ in particular, decrease to 6 - 1 0 Hz at temperatures above the thermal transition temperature for measurements at 36.4 MHz [31,32]. In this study of CDCIa solutions o f phospholipids, all linewidths were in the range 4 - 1 0 Hz, indicating relatively unrestricted motion o f the phosphate group. D. Dissociation Constant Calculations The experimental results given in tables 2, 3 and 6 were used to calculate Kd values for phospholipid/IAA interactions o f various stoichiometries. In contrast to previous work [4] it was not possible to calculate K d values under the assumption o f 2 : 1 IAA/phospholipid stoichiometry. Table 7 contains the computed Kd values for 1 : 1 interaction. Table 7 Dissociation constants (Kd) and complex shifts (A) for 1 : 1 interaction between IAA and phospholipid s.a System (IAA + . . . . . )

Probe Nucleus

Kd X 10 a (molal)

A (ppm)

DPPC DPPC PC(P.L.) PC(P.L.) PC(P.L.) PE(P.L.) PE(P.L.) PE(Sigma) PE(Sigma)

laC 31p IH laC 31p 1H 31p ~H 3,p

6+2 11 -+5 13 -+ 2 16 -+ 2 10 +-4 13 + 8 15 + 8 4+2 12 + 7

-0.89 1.34 -0.60 -0.95 1.03 -1.14 1.94 -0.70 0.83

a Values differ slightly from those in previous publication [14] in which estimated mol. wts. were used for phospholipids other than DPPC.

48

A. Marker et al., NMR studies o f horrnone (IAA).phospholipid interactions

All calculated Kd values are approx. 10 -2 molal. This is not particularly strong bind ing, though it is of the order found for some enzyme/inhibitor complexes [7]. These values are also of the same order as those found (after appropriate recalculation of K d in molal units) tbr the interaction of egg lecithin with indole and 3-methylindole in CC14[9], The changes of chemical shift on complex formation (A values) may be thought of as characteristic of and dependent on the interaction, and a reflection of the specific molecular orientation within the complex. The magnitudes of the A values are not necessarily related to the strength of the interaction K d.

IV. Discussion

An interaction occurs between the plant hormone IAA and membrane phospholipids in CDCls and the variety of NMR evidence now available makes it possible to suggest the orientation of phospholipid and IAA upon complex formation. The regions of the phospholipid molecules involved in complex formation are the -I~ (CH3) 3 or -I~H3 group, the phosphate group, and possibly the fatty acid carbonyl groups. The aromatic ring system of IAA is oriented adjacent to the -/~1(CH3)3 or -1~1H3group, and the proton on the indole nitrogen, or the carboxyl group proton, hydrogen bonds to an oxygen atom of the phosphate group. We cannot yet resolve the question of which hydrogen atom interacts with the phosphate group. In a similar system [9], involving the interaction of egg lecithin with indole and 3-methytindole in CC14, the importance of N-H...-O-P hydrogen bonding is emphasized by the large chemical shifts experienced by this proton on complex formation, and by the fact that 1-methylindole does not interact with egg lecithin. It is also possible, if hydrogen bonding occurs between the indole nitrogen proton and the phosphate group, that a second hydrogen bond is established between the indole carboxyl group proton and a carbonyl group of the fatty acids. The similarity of K d values for phosphatidylcholines and phosphatidylethanolamines may indicate that similar energetic requirements for both classes of phospholipids are involved in complex formation with IAA. This may also suggest that the phosphate group is the important binding site, with charge-induced dipole interaction between the aromatic ring system of IAA and -~(CH3)3 and -I~H3 groups providing further stabilisation for the complex. This orientation is different to that proposed by Weigl [33] for an aqueous system in which N-H.._O-P and -CO2H/-~4(CH3)3 interactions were envisaged. In addition, Weigl observed differences (in terms of solubility changes) in the consequences of IAA-phospholipid interactions, depending on the nature of the phospholipid. He ascribed the differences to differences in head groups, and gave no attention to the possible effect of differences in fatty acid composition. An important aspect of the present work is that altering the fatty acid composition of the phospholipid (e.g., from DPPC to plant source PC) alters the magnitude of the changes in chemical shifts of the 1H and 13C atoms of the choline head groups induced

A. Marker et aL, NMR studies o f hormone (1AA)-phospholipid interactions

49

by complex formation with the hormone (tables 2, 3 and 4). Similar effects due to alteration of fatty acid composition were also noted on the magnitudes of the induced 31p chemical shifts (table 6). In addition, although the results are not as clear, our data reinforce those of Weigl in that changes in the polar group (e.g., as from choline to ethanolamine) must also cause changes in the consequences of hormone binding. The behaviour of ionic compounds in CDCIa is not sufficiently similar to behaviour patterns observed in aqueous systems for the results to be easily extrapolated from on~ medium to the other. However, at least in CDCI3 it seems that the hormone can react with a range of different phospholipids, causing different effects on the different phos pholipids, depending on their precise composition. Since the phospholipid composition (in terms of different head groups and different fatty acids) is an extremely variable feature of membranes of different organs and tissues, it seems probable that complex formation between the hormone and the membranes of different tissues will produce different (or even no) effects. Thus, a biologically meaningful concept of hormone target tissues, but the induction of specific responses being determined by the precise composition of the receptor phospholipid. A mechanism such as this could operate as well as, or in conjunction with, specific interactions between hormone and intrinsic membrane proteins.

Acknowledgements We thank Dr. A. Wood for useful discussions, Mr. E.H. Williams for assistance with measurements of NMR spectra, and Mr. M. Jusaitis for determining the fatty acid composition of the phospholipids. This work was funded by Australian Research Grants Committee grants to L.G.P. and T.M.S.

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A. Marker et aL, NMR studies o f hormone (1AA)-phospholipid interactions

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