179

P|I: S0307-4412(96)00044-1

Light-induced Proton Transport through Chloroplast Membranes

JOHN W. HO and EDWINA SAU-MAN PO

Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

Introduction Environmental contamination has become a worldwide concern. Many human activities are endangering the quality of the environment and it is important to address these problems. The public is aware of the environmental damage caused by pesti- cides, halogenated hydrocarbons, nuclear radiation, toxic chemical wastes, etc.

Experiments dealing with enzyme assays for the assessment of risk are part of the biochemistry laboratory courses. Enzyme complexes such as ATPases from spinach leaves (Spinacia oler- acea L) have been used to study photosynthesis. During the process of photosynthesis, electrons are transferred from water to NADP + via an electron carrier system. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes and coupled to ADP phosphorylation. In both oxidative phosphorylation and photo- phosphorylation, electron flow through a carrier chain is linked in some way to the generation of ATP. The production of adeno- sine triphosphate (ATP) is important in maintaining biological functions. The inhibition of ATPases perturbs ATP production and cell functions. In chloroplasts protons are transported across the thylakoid membrane from the outside to the inside. A high energy proton gradient is generated by illuminating chlor- oplasts. A rise in the pH of the medium is observed. This rise in pH indicates proton uptake by the chloroplasts. The pH change is measured with a semi-micro glass pH- electrode. The relative rate of change in pH in the presence of an environmental pollu- tant can be compared with the control. Assays for these enzymes indicate the inhibitory or toxic effects of environmental pollu- tants such as pesticides on the biological activity.

In this experiment a simple method is introduced to demon- strate the formation of the proton gradient across the chlor- oplast membranes from spinach leaves. The method is applied to study the inhibitory or toxic effects of chemicals and pesticides.

Materials and methods Tricine, sucrose, acetone, potassium dibasic phosphate, sodium chloride were purchased from Sigma Chemicals (St Louis, USA). Pesticides and spinach were purchased from local super- markets. Other chemicals were of analytical grade.

0.01 M NaC1 and 2.1 mM sucrose by stirring for 30 s, or longer if needed, to produce a uniform suspension. The chloroplast preparation is stored in an ice bucket or a cooler until use. The biological activity of the enzyme complexes in chloroplasts remains stable for a few hours.

Determination of chlorophyll content The chlorophyll suspen- sion (0.05 ml) is mixed with 9.95 ml of 80% acetone in water according to the previous method. 2 The mixture is spun in a table-top centrifuge for 10 min. The supernatant is transferred to a cuvette for spectrophotometric absorbance measurement at 652 nm. The 80% acetone solution is used as the blank. The chlorophyll content is determined by the following equation:

[Chlorophyll]mg/ml = m652nm/(E: X 1)

where e: absorption coefficient for chlorophyll, 34.5 ml mg -~ cm -1, and 1: path length of solution in cuvette.

Measurement of proton activity A schematic setup for the experiment is shown in Fig 1. A trace of copper sulphate solu- tion is recommended to add into the water bath in order to eliminate IR radiation from the light source. An aliquot of chloroplast suspension (3-5 ml) at a concentration of about 300 pg chlorophyll ml is placed in the assay container or a test tube that can hold a semi-micro pH electrode. A small magnetic stir bar is needed for the uniform mixing of the solution. The pH electrode is inserted into the test tube for recording of the pH change. The initial pH of the reaction mixture is adjusted to 6.0 with diluted hydrochloric acid. The projector lamp is turned on to illuminate the chloroplasts. The pH change is traced on a chart recorder if available or the pH is recorded at a regular time interval with a timer. The light is turned off after the pH reaches a plateau. The pH drop is continuously recorded for 60 s or longer until the pH almost returns to the initial pH. The experi- ment is repeated with a fresh aliquot of chloroplast suspension with an addition of an inhibitor or a potential pollutant as desired. The proton concentration is calculated according to the previous method?

Results and discussion Paralleling the science course the biochemistry practical lab work related to environmental issues is developed. The experi- ment described has been successfully carried out by graduate students and teaching assistants. Part of the laboratory work deals with the isolation of spinach chloroplasts. The experiments are technically simple. The materials employed are relatively inexpensive. The source of the enzyme complexes are isolated from spinach purchased from a local supermarket. The pro- cedure described does not need special equipment other than those already present in most student laboratories. However, among those common buffers, such as Trizma, HEPES, Bicine, CAPS, Bis-Tris, MOPS we have tried, only Tricine buffer range

Preparation of spinach choloroplasts Chloroplasts are isolated from spinach leaves in 0.02 M Tricine-NaOH buffer, pH 8.0, containing 0.01 M NaCI and 0.4 M sucrose according to the previous method with modificationsJ The midribs from spinach leaves are discarded and the leaves are cut into small pieces into a precooled blender containing 100 ml of cold Tricine-NaOH buffer. The leaves are blended at top speed for 5-10 s. The homogenate is filtered through three layers of cheesecloth. The filtrate is run through another three layers of clean cheesecloth again. All the liquid is squeezed from the cheesecloth. The filtrate is centrifuged at 1000 xg for 2 rain. The supernatant is transferred to precooled centrifuge tubes and spun at 3000 xg for 3 min. The supernatant is discarded and the pellets are suspended in 50 ml of 0.02 M Tricine-NaOH buffer containing

i I - .

water out water in Black box

transparent lab-built watcrjackcted reaction vessel

Figure 1 Schematic setup of the appartus for measurement of pH change

BIOCHEMICAL EDUCATION 24(3) 1996

180

from 2.3 to 3.0 mM has resulted in a good, measurable signal. The concentration range is good enough for chlorophyll concen- tration as low as 1 mg per assay. Tris buffer produced a noisy signal and could not be used for a sensitive measurement. Other buffers did not work as well. An optimized sucrose solution range from 2.1 to 2.5 mM was adapted for the experiment to obtain a good measurement of the pH change. Also, an important charge balance upon illumination of the chloroplasts is required to produce a sensitive signal. Sodium chloride solu- tion (range from 0.08 to 0.13 M) in a 3-ml assay is needed to produce the one-step signal for the assay (Fig 2). A clear one- step signal is needed for the measurement of the inhibitory or toxic effects of a pollutant. Different sources of spinach have been used for the experiment. Spinach from a supermarket in the USA or in Hong Kong was used and found to be contami- nated by pesticides to a different extent. The pH change (ApH) shows the extent of contamination (Fig 2). A larger ApH is obtained from cleaner chloroplasts. Spinach received directly from a farm in the USA provides a good source of clean chlor- oplasts. However, the contamination can be reduced. Spinach leaves were immersed in clean water for about half an hour and

pH

- - 7 "

a

ApH

Time Figure 2 Light-induced pH changes in chloroplasts, (a) the control, (b) 1.3/zM N-ethylmaleimide, (c) 3.0 #M carbamate in the assay. The arrows indicate the times at which a slide projector lamp was switched on and off.

washed with distilled water prior to the preparation of chloroplasts.

In addition, coupled with the above-mentioned notion, a sen- sitive signal can be obtained by using a good light source which should be stable with a constant light intensity. A regular slide projector has served our purpose. The experiment setup was housed in a large cardboard box so that there would be very limited interference from stray UV light in the room. A good reproducible signal is readily obtained. For the undergraduate students' laboratory work, a chart recorder Model 1201 from Linear Instrument (Boston, USA) was connected to an Orion pH meter Model SA 720 (Boston, USA) for recording a small pH change. A single junction refillable semi-micro pH-electrode Model HIP (140 x 6 mm diameter) from Milano, Italy was employed for the experiment. For a better recording, a Hewlett Packard integrator Model HP3395 (Palo Alto, USA) was used. With an integrator, a more sensitive signal was obtained with less noise even when 1 mg of chlorophyll was used for the assay. A micromagnetic stirrer was used for a 3-ml assay. It was important to get a steady initial pH reading before the slide projector lamp was turned on.

The proton activity in chloroplasts can be quantitatively com- puted based on the previous method by the addition of 10/~1 of 1 M HCI to the assay; thus, the amount of proton movement is calculated based on the titration? Also, the relative rate of proton pumping activity is related to the amount of chlorophyll per assay. For more advanced study, the data on the pH change or the pH trace on the chart recorder are treated according to the previous method. 3 However, the calculation is complicated. At present, the ApH and the initial rate of proton uptake are measured for an undergraduate lab work. The measurement of the initial rate is obtained from the slope of the rise of the pH profile on the chart recorder.

In conclusion, the ATPase complexes from spinach provide a sensitive marker of potential toxicants and environmental pollu- tants including heavy metals. 4 The experiment is easy to set up for the undergraduate or more advanced study. It is an excellent practical laboratory for science students who can complete the lab work within one lab period without much difficulty.

Acknowledgements The authors are grateful to the RGC Ref no CUHK 371/95M for financial support of this work. Additional support for ESMPo was provided by the CU assistantship. We also thank P P F K Kwok and W K Yam for the initial trial of experiments.

References 1 Davenport, J W and McCarty, R E (1986) Biochim BiophysActa 851,

136-145 2 Arnon, D (1949) Plant Physio124, 1-15 3 Ho, Y K and Wang, J H (1981) J Bioenerg Biomembr 13, 5(6),

229-239 4 Carmeli, C, Tadmor, O, Lifshitz, Y, Ophir, R and Carmeli, S (1992)

FEBS Lett 299, 227-230

BIOCHEMICAL EDUCATION 24(3) 1996