The effect of dimethyl sulphoxide on electron transport in chloroplasts

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THE EFFECT OF DIMETHYL SULPHOXIDE ON ELECTRON TRANSPORT IN CHLOROPLASTS S.G. REEVES* and D.O. HALL Department of Plant Sciences, King's College 68 Half Moon Lane, London SE24 9JF, England. SUMMARY The effect of DMSO (dimethyl sulphoxide) on electron transport in chloroplast membranes has been studied. It has been found that concentrations of DMSO up to 20% (v/v) do not inhibit electron transport in freshly isolated chloroplasts, but that higher concentrations start to cause inhibition. However, in chloroplasts that have been aged for 8 to 24 hours by storage at 4'C, the addition of DMSO at concentrations up to 20% causes Possible mechanisms for this stimulation of electron transport. effect are discussed. INTRODUCTION In our laboratory we have become interested in the reactions of chloroplasts over extended time periods (Rao et al, 1976) and the storage and ageing of chloroplasts (KulandaiKluand hall, 1976). DMSO is a reagent that is known to affect phospholipid membranes, (Lyman et al, 1976) so we decided to study the effect of DMSO on chloropTZstelectron transport and on ageing in chloroplast membranes. MATERIALS AND METHODS Chloroplasts were extracted by a technique similar to that of Lilley and Walker (1974) from spinach obtained from Covent Garden Market or grown in a greenhouse. Chloroplasts were homogenised using a Polytron blender, in a medium containing 0.33M Sorbitol, 5mM MgCl and 1mM ascorbic acid, pH adjusted to 6.5. thrgugh cheesecloth, centrifuged and resuspended to a concentration of 1 mg chlorophyll/ml in a medium containing D.33M sorbitol, 2mM EDTA, 5mM MgC12 and 50mM Hepes, pH adjusted to 7:6. The chloroplasts obTained were usu';i;lly 40-80% intact, ("type A" according to Hall (1972)). experiments were carried out in 15 ml (11,000 lux white light) water bath at A 2 ml reaction mixture was used containing the reactants described in the text plus 100 ug chlorophyll and 12 umoles K Fe(CN) The reaction was terminated by the addition of 2% trqchloro acid; 2'cetic the solutions were clarified by centrifugation and the amount of K Fe (CN) reduced calculated by the decrease in absorbance at 420 am. All pates were calculated as moles K Fe(CN) reduced/mg chlorophyll/hr. Oxygen evolution experimen ? s were6carried out in a pair *

To whom correspondence

should

be addressed

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of matched oxygen electrodes (Hansatech Ltd., King's Lynn, Norfolk) essential18 as described by Delieu and Walker (1972) at a temperature of 20 C in saturating white light. The reaction mixtures were as described in the text. Measurement of hydrogen evolution in the chloroplast-hydrogenase system and in the dithionitehydrogenase system was as previously described (Rao -et -'al 1976). All available.

chemicals

were of the highest

purity

commercially

RESULTS Table I shows the effect of DMSO on light-induced electron transport in spinach chloroplasts, in a medium containing 50mM Tris, pH 8.0 and 100 pg chlorophyll. Each result is derived -from a separate experiment, carried out over a period of twelve months. Although there is considerable variation in the rates of electron transport, two types of result can be seen. Firstly, if the rate of electron transport in the absence of DMSO was high, then the addition of DMSO to the reaction mixture, up to a concentration of 20%, had little effect. Secondly, if the rate of electron transport in the absence of DMSO was low, then the presence of lo-20% DMSO stimulated electron transport. In both cases DMSO concentrations in excess of 20% usually caused inhibition of electron transport, with complete inhibition usually being caused by 60-80% DMSO. Control experiments, carried out concurrently with those shown, demonstrated that light and active chloroplasts were required for K3Fe(CN)6 reduction, and DMSO alone had no effect. The change in response of the chloroplasts to DMSO over the course of a year led us to investigate the effect of DMSO on i.e. chloroplasts stored at 4'C artificially "aged" chloroplasts, for various periods of time. Fig. la shows such an experiment. The rate of K Fe(CN)6 isola 2ed reduction was measured in aliquots of a freshly chloroplast suspension in a reaction mixture containing 50mM Tris pH 8.0, and a range of DMSO concentrations from O-60%, using the The chloroplast suspension was then illuminated water bath. stored at 4'C for 8 hours and the experiment repeated on a further After storage of the chloroplast suspension for set of aliquots. a further 16 hours at 4'C the experiment was repeated for the final time. As can be seen from Fig. la, at zero time, concentrations of 10 and 20% DMSO had little effect on electron transport, 40% DMSO caused a large inhibition and 60% DMSO caused total inhibition. However, after 8-24 hours storage, the control rates of electron transport (in the absence of DMSO) had slowed considerably, but these were stimulated by lo-40% DMSO, with 60% DMSO again causing total inhibition.

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The effect of DMSO on chloroplast electron transport. Experimental details as described in the text. The rates of electron transport are expressed as moles K Fe(CN) reduced/m9 chl/hr. The table shows the r ;! sults 8 f ten experiments carried out over the course of 12 months. (- indicates rates not determined) Rate of electron transport reduced/m9 chl/hr).

Expt.

No.

1 5 6

184 284 244 480 52 ;;

5% 248 116 44

10%

20%

292 248 -

128 248 480 220

:

30%

256 -

l3 1:

(moles

K3Fe(CN)6

% DMSO added to the reaction 0%

1 2

355

308 1;: 326

1 -

164 304 324

308 336

40%

556 276 252 76 124

mixture 50%

40 -

60%

70% 80%

0

-

-

2:;

-

-

1;8

1

;6

1 32 -

0 0 -

44 40 0 20

Figure lb shows a similar experiment carried out on a chloroplast preparation which showed stimulation of electron transport on the addition of lo-20% DMSO before any deliberate ageing. Comparison of Figs. la and lb shows that in Fig. lb there was less decrease in electron transport over the first 8 hours of ageing as if the chloroplasts were already somewhat aged on extraction. The chloroplasts used in Fig. lb were also studied in the oxygen electrode when first extracted and after 24 hours of ageing. These experiments were carried out in two different reaction mixtures. The first was 50mM Tris pH 8.0, and the second was a more complete mixture identical to the chloroplast resuspending medium. In both cases 6mM K3Fe(CN)6 was used as the electron acceptor. The effects of DMSO on the fresh chloroplasts are shown in Figs. 2a and 2b. When the chloroplasts were illuminated in a reaction mixture containing only 50mM Tris, they showed electron transport to K Fe(CN) and concomitaiit 0 evolution; this electron transgort cogld be uncoupled by2the addition of NH4C1. After about 3 minutes of illumination the rate of electron transport decreased until it stabilised at a lower level (Fig. 2, If the experiment was repeated in the presence of Trace 1). 10% DMSO a lower initial rate of electron transport occurred,

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The effect of DMSO on K3Fe(CN)6 reduction in fresh and aged chloroplasts. The rates of electron transport were measured as in the text. The chloroplasts were used immediately after gxtraction (zero hrs, x x), after 8 hrs storage at 4 C (8 hrs, o o), and after 24 hrs storage at 4'C (24 hrs, a---n). The two experiments shown were carried out on two separate chloroplast preparations.

However, which could also be stimulated by the addition of NH Cl. this rate did not slow down as quickly as the rate 14 the absence of DMSO, and stabilised at a faster rate than in the absence of (All the traces shown in Fig. 2 are from DMSO (Fig. Za, Trace 2). experiments run on matched oxygen electrodes). The traces in Fig. 2b show the experiment carried out using the more complex reaction mixture (identical to the resuspending It can be clearly seen that the same pattern emerges medium). from this experiment, except that the initial rate of electron transport is linear for a longer period of time, thus extending the time before the reaction mixture containing DMSO gives a higher rate of oxygen evolution than the reaction mixture without DMSO. (When using the complex reaction mixture the chloroplasts were osmotically shocked to allow penetration of the KSFe(CN)C. Figs. 2c and 2d show the same reaction mixture, using the As can be same chloroplasts but after storage at 4 C for 24 hours. seen, the difference between the oxygen electrode traces are much

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In Fig. 2c (reaction mixture of 50mM pH 8.0) there more dramatic. is almost no electron transport in the absence of DMSO, and considerable electron transport in the presence of 10% DMSO. In the case of the complex reaction mixture there is some electron transport in the absence of DMSO but this soon falls to zero In the presence of 10% DMSO (Fig. 2d, Trace 2) (Fig. Zd, Trace 1). the rate of electron transport is similar to that in Fig. 2c, Trace 2. The aged chloroplasts showed no stimulation by NH4Cl at Similar results could be obtained using methyl any stage. The viologen instead of K3Fe(CN) as an electron acceptor. water bath, "ageing" experiments were re 8 eated in the illuminated using the complex reaction mixture instead of 50nM Tris and using osmotically shocked chloroplasts to remove the oufer envelope and allow the K Fe(CN) to reach the thylakoids. The results were similar to z hose s Rown in Fig. la. As we were also interested in the hydrogen-evolving chloroplast hydrogenase system (Rao --' et al 1976) we studied the effect of various concentrations of DMSO on this system and the dithionitehydrogenase system. The results are shown in Figs. 3a and 3b. The hydrogen evolution in the chloroplast-hydrogenase system was linear for the 100 minutes the assays were run, with a control rate of 29 moles H evolved/mg chlorophyll/hr. As can be seen in Fig. 3a, the a ii dition of DMSO caused a linear decrease in hydrogen evolution, with 100% inhibition at 63% DMSO. A similar result was obtained in the dithionite-hydrogenase system (Fig. 3b). DISCUSSION DMSO is a well-known aprotic solvent that can reversibly unfold proteins at high concentrations presumably by affecting hydrogen bonding and hydroph0bi.c interactions. The unfolding p-ocess for a particular protein, at a particular temperature, occurs at a critical DMSO concentration, usually 60-70%, (see, for example, Cammack, 1976). It is also known as a cryo-protective agent (Lovelock and Bishop, 1959) and is thought to stabilise phospholipid membranes by decreasing membrane fluidity, as a result of increasing their phase transition temperature (Lyman -et -'al 1976). The results described in this paper show that concentrations of DMSO of lo-40% can partially reverse "ageing" of chloroplast electron transport. It should be pointed out that the observed stimulation does not increase the rates of electron transport to levels higher than can be obtained in active preparations of chloroplasts from fresh, healthy spinach leaves. The exact effects of DMSO in our systems are unknown. It is possible that the DMSO causes a reorganisation of the chloroplast membrane system, which has presumably been altered by the ageing process (Kulandaivelu and Hall, 1976). This reorganisation could then allow previously unfavourable electron transport reactions to occur.

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.

SOWW3020

-

10-

DMSO IN WE % RhcTIot4 MlXTUF

Fig.

3

The effect of DMSO on hydrogen evolution. Details as described in the text. Fig. 3a shows the effect of DMSO on a chloroplast-hydrogenase system, and Fig. 3b the effect of DMSO on a dithionitehydrogenase system.

Fig.

2

The effect of 10% DMSO on oxygen evolution in fresh and aged chloroplasts. The rates of electron transport were measured in matched K Fe(CN) as described in the text. oxygen electrodes, The react1 -a n mixt h re was the terminal electron acceptor. in Figs. 2a and 2c was 501nM Tris pH 8.0. The reaction mixture in Figs. 2b and 2d-was the complex mixture, containing 3301nM Sorbitol, 2mM EDTA, 5mM MgCl and Figs. 2Z and 2b wgre ob?ained with 50mM HEPES, pH 7.6. with freSh chloroplasts and Figs. 2c and 2d were gbtained the same chloroplasts stored for 24 hrs at 4 C. In all four figures Trace1 is the control and Trace 2 contained 10% DMSO.

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A second alternative, suggested by the oxygen electrode traces (Fig. 2) is that DMSO is preventing photodestruction of the chloroplast. The process of lipid peroxidation has long been associated with "ageing" of membranes (Wills, 1969) and this process is thought to involve singlet oxygen, superoxide or the It is possible that DMSO is acting as a hydroxyl radical. scavenger of one or more of these species (see, for example, McCord and Fridovich, 1969) thus decreasing the detrimental effect on the chloroplast membranes. The traces in Fig. 2 show that DMSO acts by preventing the decay of electron transport over a period of time, rather than by stimulating the rate. It should also be noted that electron transport in fresh chloroplasts could be uncoupled by the addition of NH Cl in the presence of 10% DMSO. has This would suggest that no exte Asive membrane re-arrangement occurred, as this would probably cause uncoupling on its own. The experiments on the hydrogen-evolving systems show simpler responses. The data in Fig. 3 show it would be possible to use the system in concentrations of DMSO up to approximately 40%, and lose only half of the control activity. This is important if parts of the system are to be replaced with analogue compounds which are soluble in DMSO (Que -et -'al 1974 and Holm, 1975). The effect of DMSO on chloroplast electron transport, as shown in this paper, has implications for long term use and storage of chloroplasts. Various aspects of this problem are currently under study in this laboratory. ACKNOWLEDGEMENTS The authors would like to thank discussions. This work was supported under contract number OZO-76-ESUK.

Dr. B. Halliwell by the European

for helpful Commission,

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in microsomes.

of

of