Mobility of Spin Probes in Solution and in the Chloroplast Membrane Surface

Mobility of Spin Probes in Solution and in the Chloroplast Membrane Surface D. KAFAUEVA, M.

BUCHEVA

and E.

ApOSTOLOVA

Bulgarian Academy of Sciences, Central Laboratory of Biophysics, Acad. G. Bonchev Sec, Bl. 11 Sofia 1113, Bulgaria Received June 3, 1985 . Accepted July 23, 1985

Summary The absolute value of solvent bulk viscosity is determined using rotation correlation time of spin-labelled aminoadamamane and spin-labelled pelargonic acid as a sensitive parameter. The alteration of the viscosity of the medium at different temperatures could also be successfully measured with both spin probes. lntact chloroplasts are labelled with the same spin probes. The distribution of the spin probes is such that: 30% are situated in the chloroplast envelope, 60% in the thylakoid fractio n and o nly 10% in the cytoplasm. The spin probes aminoadamantane and spin-labelled pelargonic acid are embedded in the chloroplast membranes and the nitroxyl radical is located in the hydrophyJic part of the membrane near its surface. The microviscosity in this region is aboU[ 3 mPa.s. These spin probes are used to determine alteration of the membrane microviscosity over the temperature range 243 °K_303 OK.

Key words: Chloroplast membrane surface) microviscosity. spin probes. viscosity ofsolvent.

Introduction The viscosity of the chloroplast thylakoid membranes plays an imponant role in controlling the light reactions during the process of photosynthesis (Quinn and Williams, 1978; Barber et aI., 1980). For this reason the determination of the viscosity of the membrane is important. The viscosity is usually studied by ESR or fluorescence spectroscopy. The sensitivity of the ESR spectral parameters to the viscosity of the solution is theoretically evaluated and discussed in a large number of papers (Likhtcnshtein, 1974; Axel, 1976). When nitroxyl radicals are incorporated into biological membranes or lipid bilayers, theoretical difficulties arise in determining the correct relationship between ESR spectral parameters and the viscosity of the nitroxyl radical environment. This may be due to the heterogeneity of this environment as well as to the unknown mechanism of the motion and reorientations of spin probes. This is Abbreviation list: An - hyperfine coupling constant, Tc - rotation correlation time, ASL 3-[/ 1'-adamantyll-carbamoyIJ-2,2,5,5-tetramethylpyrolidin-l-
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why the term microviscosity has been introduced and related to the mobility of spin probes in ordered systems. Empirical correlation between the experimental parameters of ESR spectra and microviscosity of the native membrane and lipid bilayer has been established (Wu and McConnel, 1975). More often microviscosity has been evaluated from the same ESR spectral parameters (rotation correlation time and hyperfine coupling constant) which are used for determination of viscosity of solutions (Berg et al., 1979). In this study the values of rotation correlation time (T,) and hyperfine coupling constant (Ao) of spin-labelled aminoadamantane and spin-labelled fatty acids (pelargonic, stearic, and palmitic) embedded in chloroplast membranes are compared with the same values in ethanol or glycerol~water mixtures in parallel experiments. The aim is to obtain more information about the viscosity of the chloroplast mem~ brane. The actual localization of spin probes in chloroplasts is also tested. The sensitivity of rotation correlation time, Tc of spin~labelled aminoadamantane to the viscosity of the solution is also discussed in this study.

1

, 2

Fig. 1: The 9.5 GHz ESR spectra of PLSL in intact chloroplasts. (1) Typical ESR spectrum recorded at room temperature with well separated. lines without splitting; instrument set as in Materials and Methods, microwave suppression 30 dB. (2) Spectrum of frozen sample, recorded at 223 OK, microwave suppression 24 dB, represented only for comparison with fast rotating spin probes in the temperature interval studied. here.

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6Gs

>-----<

Fig. 2, ESR 'peelra ofPLSL and SSL in ethanol and chloroplasts. (\) PLSL in ethanol at 293 oK, 18dB microwave supression; (2) PLSL in ethanol at 273°K, 24dBj (3) PLSL in ethanol at 253 oK, 30dB; (4) SSL in ethanol at 253 oK, 18dB; (5) PLSL in chloroplasts at 293 oK, 30 dB; (6) PLSL in chloroplasts at 263 oK, 24 dB.

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Materials and Methods Fatty acids were labelled according to the procedure of Rozantsev (1978) and the following spin probes were obtained: 2,2,6,6-tetramethyl+Oxypelargonylpiperid.l-oxyl (PLSL), 2,2,6,6tetramethyl+oxypalmitoylpiperid·l-oxyl (PASL) and 2,2,6,6-tetramethyl+oxysteranoylpi. perid.l-oxyl (SSL). Spin-labelled aminoadamantane (3..[/l'-adamantyl/-carbamoyl}2,2,5,5-tetramethylpyrolidin-l~xyl [ASL] was synthesized from aminoadamantane and 2,2,5,5,-tetramethylpyrolin-3carboxyl·l-oxyl. ESR spectra were recorded on a ESR-220 spectrometer. The temperatures in the capillary pipette, containing the sample, were controlled by a nitrogen gas-flow temperature regulator. They were measured using a copper-constantan thermocouple inserted in the cavity with a maximum relative error ± 1.0 oc, The spectra were recorded at 9.05 GHz with 27 min scan time over 200 Gs, magnetic field 3440 Gs, time constant of 0.5 s. The rotation correlation time Tc was calculated as in (Kafalieva and Apostolova, 1984). Hyperfine coupling constant (An) was determined as a splitting between the central line and higher field resonance line of the spectra (Likhtenshtein, 1974). Intact pea chloroplasts were prepared by a method described in (Leech, 1964) and thylakoids as in (Argyroudi-Akoyunoglou and Castorinis, 1980). The spin probes (10-4M) were introduced into chloroplast membranes by a solution of ethanol, so that the final ethanol concentration was always less than 1 %. The chloroplasts were thoroughly mixed with spin probes for 60 min for ASL and PLSL, and 120 min for PASL and SSL. They were washed three times with buffer. The spin probe concentration is calculated by the area under the ESR spectra. The viscosity ('1) of the solutions was measured. with a routine viscosimeter and compared with the values given in the reference book.

Results and Discussion All spin probes show three resonance line spectra without splitting over the temperature interval studied in this work (Fig. 1 and Fig. 2).

Spin probes in solution Rotation correlation time values of spin-labelled aminoadamantane dissolved in solutions of different viscosity at 293 OK are presented in Table 1. The values are the average of five independent experiments. In Fig. 3, Tc values are plotted against the viscosity of the solvent in which ASL is dissolved. The solvents are the same as listed in Table 1. The rotation correlation time Tc of ASL, as it is demonstrated in Fig. 3, increases linearly with the increase of the viscosity. Linear dependence gives the possibility to obtain the unknown absolute viscosity value of a solvent or a mixture. The variation of the viscosity of a solvent caused by temperature changes reflects the motion of the spin.labelled molecules. The dependence between T, and ~/T for ASL and PLSL in absolute ethanol are presented in Fig.4. The linear relationship between T, and ~/T supports the assumption of Brownian diffusion mechanism of motion and applicability of the Stokes-Einstein equation:

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10 0

9

8 7

,,~

6

'\2

,

5

'""

0

4 0

3

2

-~--'

2

3

45 6 i- 8

10

9

'1 ImPas 1 Fig. 3: The rotation correlation time Tc of ASL in solvents of different viscosity at room temperature. The solvents were selected with viscosity 0.5-10mPa.s (see Table 1). The values of Tc are the average of five independent experiments. Table 1: The rotation correlation time Te of ASL in solvents with different viscosity. Solvent

Viscosity mPa.s

TeX 10 -

methanol water ethanol 20% glycerol butanol 50% glycero l octanol

0.59 1.00 1.20 1.73 2.95 6.05 10.69

0.70 1.10 1.30 1.57 3.20 6.27 9.50

10

s

rdrX 10 -

10

m

5.1 4.6

4.7 5.2

4.7 4.7 5.7

Values of the viscosity arc obtained fro m the reference book, 4

1['

~rr

T, - --~

3 KT

This equation gives the opportunity to determine the effective radius of the molecules. The effective radius of ASL obtained in the solutions studied are presented in

Table 1. ESR spectra of PLSL are similar to those of ASL and also show linear dependence between T, and ~/T (Fig. 4). Different slopes of the curves in Fig. 4 emphasize differ-

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D. KAFALIEVA, M. BUCHEVA and E. APOSTOLOVA 15

ASL

13 11

'"

0-

9

'T

Q X

7

~

5

3

33

~2

10.05

15.8

if [m~asJ

30.08

Fig. 4: The rotation correlation time 7"e of spin probes ASL (--e--) and PLSL ( - -0 - - ) in ethanol plotted against 'J/T. The curves are obtained from experimentally determined values of Tc. T - temperature of solvent in experimental cuvette (T is ranged from 223 OK to 303 OK) and 71 - the viscosity at the corresponding temperature. ent sensitivity of the spin-labelled molecules to the viscosity of the medium. The distinction between the two curves (especially with viscosity higher than 3 mPa.s) is probably caused by the shape rather than by the nature of the spin-labelled molecules. Dependence of Te of ASL in ethanol and glycerol-water solution, on temperature are presented in Fig. 5. All Arrhenius curves are linear. The spectra and Arrhenius plots of spin probe PLSL are almost the same as those of ASL (data not shown). The activation energy for processes of reorientation obtained from Arrhenius curvesfor ASL is: in ethanol - 4,41±0.18 kcaVmol; in 20 % glycerol - 4,4±0.5 kcaV mol; in 50 % glycerol - 4.7 ± 0.5 kcaV mol. Palmitic and stearic acids attached to the nitroxyl radical gave spin-labelled molecules of a cylindric shape. For molecules with cylindrical symmetry, isotropic and anisotropic reorientation is possible. The results of spectral computation in the region of fast tumbling show that both models of reorientation are equally applicable (Axel, 1976). All spin probes rotate very fast at the temperatures studied here. For this reason the same approach for all spin probes including ASL and PLSL are used. Arrhenius curves for SSL (Fig. 4) in ethanol and glycerol - water solutions are also

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temperature I'C) 10 0 -10 -20 -30

;05

Vi

2.0

eo 15 toY" 3' 1.0 0.5

A

L5

20 Vi 1 5 c

~ ......

0.5

B

2.5 2.0 Vi 15 c

<-"to go

Fig. 5: Arrhenius plots of rotation diffusion parameter (log Tc plotted against T-l) of ASL (A) and SSL (B) in ethanol (--e--), 20 % glycerol ( - -0 - ) and 50% glycerol (A- -). Arrhenius plots of Tc of ASL ( • )and PLSL ( - - 0 - ) in chloroplasts (C).

0.5

C

~33;--~3"'5-- fl 39

4.1

10 3fT I K-' )

linear over the temperature region 243 °K-303 OK. PASL Arrhenius plots are very similar to those of SSL (data not shown). The activation energies for reorientation of SSL obtained from these plots are 3.2±0.5 kcal/mol in ethanol and 4.5±0.5kcal/mol in 20% glycerol solution.

Spin probes in chloroplasts Washing procedure of spin-labelled chloroplasts removes spin probes present in the suspension but not integrated with chloroplasts. This is confirmed by the experimental fact that ESR spectra of spin-labelled chloroplasts remained unchanged in the shape of line and amplitude, after double washing. The question where the probes are situated, in chloroplast membrane or in cytoplasm, remains open at the moment. An implicit assumption exists that such relatively hydrophobic spin probes do not persist in the cytoplasm or in the thylakoid

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matrix (Herring and Weeks, 1979). Tichonov and Ruuge (1978) supposed that labelled fatty acids penetrate the hydrophobic interior of the chloroplast membranes. It is clear that further evidence is indispensable to clarify the location of spin probes in chloroplasts. In our experiments 94 % - 96 % of the chloroplasts retained an intact envelope when isolated according to Leech (1964). Such intact chloroplasts were spin-labelled and washed twice to remove spin-labelled molecules which were not incorporated in the chloroplasts. Removal of the chloroplast envelope of the spin-labelled chloroplasts caused a decrease in the amount of the spin probes by 30 %. Chloroplasts without an envelope were obtained. according to the method of Papageorgiou and Demostenopoulou-Karaoulani (1982). The thylakoid fraction (see Materials and Methods) isolated from spin-labelled chloroplasts contained 60 % of the spin probe of intact chloroplasts. These results demonstrate that spin probes studied in this work pass through the chloroplast envelope and only 30 % of them are captured from the envelope membranes. A great part (57%-60%)" of the spin probes penetrates into the thylakoid membrane and only 10 % is situated in the cytoplasm. A comparison between An values in solvents and in chloroplasts shows that the environment of the spin probes in the membrane is hydrophylic (Table2). The data obtained with the paramagnitic expander K,Fe(CN), support the idea that nitroxyl radicals are disposed close to the membrane surface. Table 2: Values of hyperfine coupling constant (An, Gs) in solution with different viscosity and chloroplast membranes. --------------~---------------

Spin probes

Solution

ASL

PLSL

water ethanol

15.4 16.0 16.0 16.2 16.2 16.4

16.8 15.9

20 % glycerol 50 % glycerol

chloroplasts thylakoids

16.6 16.8 16.5

PASL

SSL

17.2 17.6 17.7 17.8 17.6

17.2 17.6 17.8 18.0 17.4

When 120 mM K,Fe(CN), is added, the integrated signal intensity of spin-labelled intact chloroplasts decreases by 15%-20%. It is well known that K,Fe(CN), does not penetrate into the chloroplast envelope (Heber and Santarius, 1970). The reduction of the signal comes from the spin probe in the outer envelope membrane which has close contact with K,Fe(CN),. If 120 mM K,Fe(CN)6 is added to the suspension of osmotically shocked chloroplasts, ESR signal decreases by 75 % - 80 %. In this case K,Fe(CN), can interact with spin probes in the OUler part of the thylakoid membrane and the cytoplasm. According to Berg et al. (1979), K,Fe(CN), leaks very

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slowly into the interior of the thylakoids. This shows that spin probes from the inner part of the thylakoid membrane give rise to remaining signal. These results are in good agreement with the data from the distribution of the spin probes described above. The conclusion could be made that spin probes such as ASL and PLSL are situated in the chloroplast membrane near the surface, in the hy~ drophylic region, tumbling fast and isotropically. ESR spectra of ASL in the chloroplast membrane are similar to those of ASL dissolved in 20 % glycerol (Fig. 5). In both cases T, has a values of 0.4 ns and hyperfine coupling constant An = 16.7 Gs. The similarity is extended over Arrhenius plots (Fig. 5). Arrhenius plots support the presumption of Brownian mechanism of motion. Each profile of the plots (Fig. 5) exhibits a break in the slopes at 277 OK. The activation energies in this case are in the range of 4.2-5.2kcaVrnol. Taking into consideration the similarity of the spectral parameters of ASL and PLSL in 20 % glycerol~water solution and in chloroplasts at 293 OK, it can be as~ sumed, that the viscosity of the chloroplast membrane surface is about 3 mPa.s. The local viscosity of chloroplast membrane is determined to be about 7 -10 mPa.s is if SSL is used as a spin probe. This result does not differ significantly from the data obtained by Berg et al. (1979) about the viscosity in the internal aqueous compartment of spinach thylakoids. Our results confirm the conclusion of Morse et al. (1982) that adamantane spin probe is very sensitive to events at the membrane~water interface and show the alterations of microviscosity in this region.

References ARGYROUDI-AltOYUNOGLOU, J. H. and A. CASTORINIS: Specificity of the chlorophyll-tD-protein binding in the chlorophyll-protein complexes of the thylakoid. Arch. Biochem. Biophys. 200, 326-335 (1980).

AXEL, F. S.: Biophysics with nitroxyl radicals. Biophys. Struct. Mechanism. 2,181-218 (1976). BARBER, J., W. S. SHOW, C. SCONFFLAIRE, and R. LANNOGE: The relationship between thylakoid staking and salt induced chlorophyll fluorescence changes. Biochem. Biophys. Acta 591, 92 - 103 (1980).

BERG, S. P., D. M. LUSCZAKOSKI, and P. D. MORSE: Spin label motion in the internal aqueous compartment of spinach thylakoids. Arch. Biochem. Biophys. 194, 138-148 (1979). HEBER, U. and K. A. SANTARlUS: Transfer of ATP and ADP across the chloroplast envelope. Z. Naturlorsch. 25 b, 718 -728 (1970). HEltRlNG, F. G. and G. WEEKS: Analysis of Dictyostelium discoideurn plasma membrane fluidity by electron spin resonance. Biochim. Biophys. Acta 552, 66-77 (1979). KAFALlEVA, D. and E. APOSTOWVA: Composition and function of chloroplast and thylakoid membranes affected by digitonin and glutaraldehyde. Advances in Photosynthesis Research 3,39-42 (1984).

LEECH, R. M,: The isolation of structurally intact chloroplasts. Biochim. Biophys. Acta 79, 637-639(1964).

LIKHTENSHTEIN, G. I.: Spin labeling method in molecular biology, John Wiley and Sons, New York, London, Sydney, Toronto, 1974. j. Plant P/rysiol. Vol. 122. pp. 445 - 454 (1986)

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MOIlSE, P. B., D. M. LUSCZAIOSXI-NESBm, and R. B. Cu.nsEN: Adamantyl nitroxide. A spin label for probing membrane surfaces. Chern. Phys. Lipids 31, 257-273 (1982). PAPAGEORGIOU, G. C. and E. DEMOSTENOPOULOU-K.uAOULANI: Stabilization of the morphology and photosynthetic function of isolated chloroplasts with GA. Z. Pflanzenphysiol. 105, 201-210 (1982). ROZANTSEV, E. D.: Free nitroxyl radicals, Plenum, New York, 1978. QUIN, P. J. and W. P. WIWAMS: Plant lipids and their role in membrane function. Prog. Biophys. Mol. 34, 109-173 (1978). TICHONOV, A. N. and E. K. RUUGE: ESR study of electron transport in photosynthetic systems. Molecular Biology (in Russian) 12, 1028-1038 (1978). Wu, S. H. W. and H. M. McCoNNEU.: Phase separation in phospholipid membranes. Bi
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