[4] Ion transport (H+, K+ Mg2+ exchange phenomena)

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charcoal (Norit) and 15 g o f Celite filteraid are stirred in, and the resulting suspension is filtered on a Biichner funnel. T h e mixture o f charcoal, filteraid, and sodium chloride on the filter is washed with 200 ml o f hot absolute alcohol. T o the combined filtrate and washings are a d d e d 230 ml of glacial acetic acid. (A few drops o f the resulting mixture when greatly diluted with water should give a p H o f 5.0 +-- 0.2). T h e acidified filtrate is allowed to stand at 00-4 ° overnight and the precipitate is filtered off on a Biichner funnel. T h e precipitate is then washed with 500 ml o f 95% alcohol (room t e m p e r a t u r e ) and r e s u s p e n d e d in 1.5 liters o f 95% alcohol to which has been a d d e d 300 ml o f H20. T h e resulting solution is heated to 75 °. Twenty-five grams of Norit and 25 g o f Celite are a d d e d and the hot suspension is filtered t h r o u g h a Biichner funnel p r e p a r e d with filteraid. T h e filter is washed with 250 ml o f boiling 95% alcohol. T o the combined filtrate and washings are a d d e d 1.5 liters o f hot absolute alcohol. T h e solution is cooled slowly with continuous stirring to 5 ° then left overnight at 00-4 °. T h e precipitate is filtered off on a Btichner funnel, washed with 300 ml o f 95% alcohol, and r e s u s p e n d e d in 2.5 liters o f 95% alcohol. T h e suspension is heated to 70 °, and water is a d d e d very slowly and cautiously until the precipitate just dissolves. T h e solution is then cooled slowly with continuous stirring to 5 ° and left overnight at 0°-4 ° . T h e final precipitate is sucked dry on a Biichner funnel with a plastic seal over the surface. Yield 280-375 g (60-80% on the basis o f the sodium chloroethanesulfonate).

[4] Ion Transport (H +, K +, Mg ~+ Exchange Phenomena) By RICHARDA. DILLEY Ion transport, particularly o f H + ions, has been shown to be intimately involved with electron transport and the e n e r g y conversion system o f chloroplasts. 1-3 Most o f this work derived its impetus f r o m the Mitchell chemiosmotic hypothesis, which assumes that chemical potentials o f ion gradients could be the driving force for phosphorylation. 4 1A. T, Jagendorf and E. Uribe, Brookhaven Symp. Biol. 19, 215 (1966). 2M. Schwartz,Nature (London) 219, 915 (1968). aR. A. Dilley, in "Progress in Photosynthesis Research" (H. Metzner, ed.), Vol. III, p. 1354. H. Laupp, Tiibingen, 1969. 4p. Mitchell,Biol.Rev. Cambridge Phil. Soc.41,445 (1966).

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The basic observations have been that light-induced electron flow results in H + ion uptake into the grana membrane of chloroplasts, 1 usually in exchange for K + and Mga+ ions. 5 The cation exchange for protons has not been studied in great detail, but it is known that the efflux processes are variable in circumstances where the proton uptake is consistently observed to be fairly constant in rate and extent. 5"6 The. purpose of this article is to outline the experimental techniques used in measuring these ion transport processes.

Measurement of pH Changes in Chloroplast Suspensions

Preparation of Chloroplasts. Chloroplast preparation may be carried out by a variety of methods, all giving basically the same type of proton uptake activity. 2"5'0 The routine preparation consists of grinding about 50 g of deveined spinach leaves for 30 seconds in a Waring Blendor with 100 ml of 0.4 M cold sucrose (or sorbitol) containing 0.01 M KCI, 0.02 M tricine-NaOH, pH 7.8, and 3 mM Na ascorbate. The brei is strained through at least eight layers of cheesecloth, and the filtrate is centrifuged for 1.5 minutes at 200 g. The pellet is discarded and the supernatant solution is centrifuged for 10 minutes at 1000 g. The resulting pellet is resuspended in about 20 ml of the grinding medium and centrifuged again at 1000 g for 5 minutes. The pellet is resuspended in a medium consisting of 0.4 M sorbitol, 0.01 M KC1, 3 mM Na ascorbate, and 9 mg of bovine serum albumin per milliliter. Chlorophyll is determined in the standard way? Apparatus. The recording of pH changes can be carried out using one of a variety of commercially available pH meters connected to an appropriate recorder. All that is required is that the pH meter-recorder pair provide full-scale deflection for pH changes of 0.03 to 0.3 pH unit. Depending on the buffering capacity of the reaction mixture, the light-induced pH change can be as large as 0.4 pH unit, i.e., from an initial pH of 6.0 to a pH of 6.3 or 6.4 in the light. Because temperature changes result in about a 0.02 pH unit decrease per degree of increase, the reaction should be carried out in a jacketed and thermostatted vessel. It is important to stir the reaction mixture so that ion diffusion is not unduly complicated by unstirred layer effects. Stirring also aids uniform illumination. Magnetic stirrers have been found not to introduce noise into pH recording. Illumination can be provided by a variety of light sources, such as a high intensity microscope light. Near saturating white light intensity 5R. A. Dilley and L. P. Vernon, Arch. Biochem. Biophys. 111,365 (!965). OA. R. Crofts, D. W. Deamer, and L. Packer, Biochim. Biophys.Acta 131, 97 (1967).

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for a reaction mixture containing 20 t~g of chlorophyll per milliliter is about 2 x l0 s ergs cm -2 sec -1 with the infrared radiation removed by a Corning 1-69 filter. The infrared filter (or 5 cm thickness of water containing a trace of copper sulfate) serves to alleviate most of the heating effects of the light beam. Reaction Mixture. A standard reaction mixture contains the following components in a total volume of 10 ml: 100 mM KC1, 5 mM MgCI2, 20/~g chlorophyll per milliliter as chloroplasts, and 15/J,M pyocyanine or phenazine methosulfate as the electron transport cofactor. [Methylviologen (0.4 mM) can serve in place of pyocyanine, as can trimethylhydroquinone (0.13 mM).] The pH should be adjusted to around pH 6 with standard acid or base. The light-induced pH changes can be observed at pH values from about 5.5 to 8.5. An initial pH of around 6 is a convenient pH. A typical trace for the light-induced pH changes of chloroplast reaction mixtures of the type described above is shown in Fig. 1. The rate and the extent of the pH changes may be estimated from the pH deflection due to the addition of standard acid or base. Recently Polya and Jagendorf 7 showed that chloroplast suspensions have a greater buffering power in the light than in the dark, although in my experience this is observed only with reaction mixtures of very low buffering capacity. For a variety of reasons, it is advisable to have a buffer concentration of about 0.3-1 mM in the reaction mixture. Interestingly, the response time for detecting changes in pH is markedly increased by the presence of such buffer concentrations. The reason, as first pointed out by Mitchell, has to do with the effect of buffering compounds on the rate of propagation of

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FIc;. I. Light-induced pH changes observed with chloroplast suspensions. T h e reaction mixture consists of 100 mM KCI, 5 mM MgClz, 20 ~g of chlorophyll per milliliter as chloroplasts, and 15 ~M pyocyanine. T h e initial pH was about 6, and o t h e r conditions were as mentioned in the text. 7G. M. Polya and A. T. Jagendorf, Biochem. Biophys. Res. Commun. 36, 696 (1969).

[4]

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FIG. 2. pH changes observed at pH 8 in the presence and the absence of an ATPforming system. The reaction mixture was similar to that given in Fig. I with the following additions: Curve A, 0.8 mM ADP, and 0.8 mM K2HPO4; initial pH 8.0. Curve B, the reaction mixture was similar to that for curve A except that K~HAsO4 replaced K2HPO4. In the case of curve B a typical light-induced proton uptake reaction is obtained, leading to a steady state level of proton accumulation. In curve A, steady-state pH changes are not obtained owing to the continued pH increase due to esterification of ADP with phosphate. The ongoing pH changes obtained in the case of curve A may be used as a measure of the rate of ATP formation. [M. Nishimura, T. Ito, and B. Chance, Biochim. Biophys. Acta 131, 97 (1967).

ApH from the bulk solution to the vicinity of the glass electrode. 8 The initial rate of pH change may be 3-5 times faster with 1 mM tricine present in a standard reaction mix (at an initial pH near 8) compared to the rate in the absence of buffer. The KC1 used as a suspending medium does not substitute for the buffer in this effect. In addition to measuring the reversible light-induced hydrogen ion uptake of chloroplasts, this technique is also readily used to measure the rate of photophosphorylation in chloroplasts. 9 For this assay, in a d d i t i o n to t h e a b o v e c o m p o n e n t s o f t h e r e a c t i o n m i x t u r e , a r e a d d e d the following: 1 m M ADP a n d 3 m M potassium or sodium phosphate; the p H o f this system is a d j u s t e d to n e a r p H 8. I f the c h l o r o p l a s t s a r e c a p a b l e o f c a r r y i n g o u t p h o t o p h o s p h o r y l a t i o n , the type o f p H trace o b t a i n e d is t h a t s h o w n i n Fig. 2A. A g a i n , the r a t e c a n be d e t e r m i n e d b y ap. Mitchell and J. Moyle, Biochem.J. 104, 588 (1967). 9M. Nishimura, T. Ito, and B. Chance, Biochim. Biophys. Acta 59, 177 (1964).

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a comparison to the pH change noted upon addition of standard acid or base. Under these conditions, it is difficult to evaluate how much of the initial pH change observed upon illumination is due to the hydrogen ion pump and how much reflects ongoing photophosphorylation. To check this point, one can substitute arsenate in place of phosphate and obtain a trace of the type shown in Fig. 2, curve B. In this case, since there is no net (phosphate) esterification, the only pH changes observed are those due to reversible hydrogen ion uptake activity.

Measurement of Potassium, Sodium, Magnesium, and Calcium Transport

AtornicAbsorption Spectrophotometry As stated above, hydrogen ion uptake activity in chloroplasts is usually accompanied by the extrusion of either potassium or magnesium. (In some cases, sodium and calcium may also be exchanged outward for hydrogen ions taken up.) Atomic absorption spectrophotometry is the most convenient method for measuring the concentrations of these cations, and typical measurements are described in detail by Dilley and Vernon. 5 The technique is very straightforward and will not be discussed here. Rather, various methods to separate chloroplasts from the suspending medium are discussed, thereby allowing assays to be performed on the supernatant solution. Millipore Filter Technique. This technique consists simply of passing the chloroplast suspension over a Millipore filter and thus separating the chloroplasts from the suspension. For this, various types of Millipore filters and filter holders are available. One convenient type, called the Swinny Adapter, readily fits on the end of standard syringes, and the suspension is filtered through the Millipore filter in less than 2 seconds. Larger filters can be used with a Biichner funnel to reduce the pressure below the filter and draw the suspension onto the filter. It is convenient to use the glass prefilters over the filter proper so as to reduce the probability that the chloroplasts will clog the filter holes. Millipore filters with a pore size of 0.45 ~zare very effective in separating chloroplasts from a suspension. Aliquots of the filtrate may then be treated according to the directions for atomic absorption spectrophotometry. Comparison of the data to that of standard curves of various salt solutions will allow one to determine the concentration of the given ion in the filtrate. Centrifugation Technique. If rapid separation of the chloroplasts from the suspending medium is not necessary or if Millipore filters are not available, centrifugation may be used to separate chloroplasts

[4]

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from the suspension. The resulting supernatant fluid can then be handled as outlined above for the Millipore filter filtrate. The ion content in the chloroplast fraction may be assayed following digestion of the organic matter with nitric acid, after which one suitably dilutes the digestate with distilled water. Ion Specific Electrodes f o r K + and N a +

In some circumstances concentrations of potassium and sodium ion can readily be measured with electrodes in a manner similar to the measurement of H + ion concentration. TM Electrodes made from special glass show relative selectivity, in some cases with high potassium sensitivity and lower sodium and other monovalent cation sensitivity; other glasses are available with high sodium sensitivity and relatively lower potassium and other monovalent cation sensitivity. Most of these electrodes are also quite sensitive to hydrogen ion concentration changes. The apparatus used in measuring ion concentration with the cation electrodes is similar to that used for measuring hydrogen ion concentration. The reference electrode can be the same reference electrode as that used with the pH measurement, and the cation electrode may be plugged into the pH meter in place of the hydrogen ion glass electrode. The potential generated by the cation electrode is a log function of cation concentration; hence much greater sensitivity is obtained if the concentration of the cation to be measured is fairly low in the suspending medium. In the case of chloroplast potassium efflux, it is difficult to detect the potassium efflux if the ambient concentration is greater than about 5 × 10-4 M. For this reason, chloroplasts should be prepared in the absence of either sodium or potassium ions and also resuspended in the absence of these ions. A typical reaction mixture, which we have used with good success for measuring light-induced potassium efflux, consists of the following: 200 mM sucrose, 20 mM Tris'HCl, and an electron transport cofactor of the type mentioned above in the discussion on hydrogen ion measurements. A typical experimental result for lightinduced potassium efflux from chloroplasts is shown in Fig. 3. The lack of potassium reentry into the chloroplasts after the light was turned off is attributed to a competition between the Tris cation, which is at a much higher concentration, and the K + ion, which is extruded into a low K + medium. Many types of ion selective electrodes are now becoming available 1°C. Moore and B. Pressman, Biochem. Biophys. Res. Commun. 15, 562 (1964).

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TIME (SEC) FIG. 3. Light-induced K + efflux in exchange for H + influx with spinach chloroplasts. K + concentration changes were measured with a cation sensitive electrode as described in the text. T h e reaction mixture contained 200 mM sucrose, 20 mM Tris.HCl, pH 6, and 0.13 mM trimethylhydroquinone. T h e final volume was 10 ml, and other conditions were as described in the text.

commercially, including magnesium, calcium, chloride, and many others. Information on these various electrodes is readily available from the manufacturers.

[5] Techniques for Studying Light-Induced Paramagnetism in Photosynthetic Materials by Means of Electron Paramagnetic Resonance Spectroscopy By ELLEN

C. WEAVER a n d

HARRY E. WEAVER

Introduction and Scope This article is concerned with methods relevant to performing electron paramagnetic resonance (EPR) experiments on light-induced resonances in photosynthetic materials. To this end, we consider the experimental aspects of working with aqueous suspensions of necessarily complex systems: algae, chloroplasts, subchloroplast fractions, bacteria, chromatophores, and "reaction-center" preparations. The emphasis is on experiments at ambient temperature. Techniques for quantitative and qualitative determination of the information content