Kinetic analysis of light induced proton dissociation and association of bacteriorhodopsin in purple membrane fragments under continuous illumination

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Kinetic analysis of light induced proton dissociation and association of bacteriorhodopsin in purple membrane fragments under continuous illumination
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  journal of MEMBRANE SCIENCE ELSEVIER Journal of Membrane Science 104 (1995) 65-72 Kinetic analysis of light induced proton dissociation and association of bacteriorhodopsin in purple membrane fragments under continuous illumination Meral Yticel a.., Baker M. Zabut b, [nci Erof~lu c, Lemi Ttirker d a Department of Biology, Middle East Technical University, Ankara 06531, Turkey b Education College, Directorate of Education, Gaza via, Israel Department of Chemical Engineering, Middle East Technical University, Ankara 06531, Turkey a Department of Chemistry, Middle East Technical University, Ankara 06531, Turkey Received 1 August 1994; accepted in revised form 6 January 1995 Abstract In the present work the photo responses of bacteriorhodopsin in purple membrane fragments were studied under continuous high intensity light illumination in different ranges of pH, temperature and ionic strength. The activity of bacteriorhodopsin was measured as pH vs. time by using a combined pH electrode. The activity of the bacteriorhodopsin in purple membrane fragments at acidic pH was found to be more sensitive to temperature changes than it was at physiological or higher pH values. The activity of bacteriorhodopsin was also found to be the function of varing concentrations of different salts. The kinetic analyses of light reactions (proton dissociation) and dark reactions (proton association) were carded out at different pH, temperature and ionic strengths. The kinetic data revealed that the proton dissociation in the light and proton association in the dark followed the first order kinetics in the pH range of 6.7-7.2 at room temperature. Beyond these limits the kinetic behavior of bacteriorhodopsin was found to be much more complex. Keywords: Bacteriorhodopsin; Halobacterium halobium; Proton pump ; Purple membrane fragments 1. Introduction Bacteriorhodopsin (BR) is a light energy transduc- ing retinal protein found in the purple membrane (PM) of Halobacterium halobium [ 1 ]. BR is the only protein present in PM fragments isolated in pure form (MW 26 000) and its topological structure in the membrane has already been determined [2--6]. In living cells BR acts as a light driven proton pump so that under illumination it pumps protons from the interior of the cell to the surrounding medium thereby ~' Corresponding author. 0376-7388/95/ 09.50 © 1995 Elsevier Science B.V. All fights reserved SSD10376-7388(95)00012-7 generating a transmembrane proton gradient. Halobac- terium halobium makes use of this proton gradient for the synthesis of ATP hence BR behaves as a light trans- ducer for the conversion of light energy into chemical energy. The light dependent proton transport system of BR is coupled to the photochemical conversion of the retinal chromophore of BR and the nature of the pho- tocycle. Although, extensive amounts of experimental research on the photochemical reactions of BR has been accumulated over the last two decades [7-11 ], due to the complexities existing in the kinetic behavior of the intermediates of the reaction cycle, the general features  66 M. Yacel et al. / Journal of Membrane Science 104 (1995) 65-72 of the proton transport mechanism occurring in BR are not well-understood yet [ 12]. BR in PM fragments isolated from Halobacterium halobium, has both fast and slow phototosensory prop- erties [ 13]. These properties can be used by incorpo- rating PM fragments into biosynthetic membranes thus forming intelligent materials. Fast photosensory prop- erties of BR could function in short illumination time. Potential application areas were reported as laser eye protection systems, optical sensors and halographic films. In those studies the researchers have simplified the photocycle for explaining of the behavior of BR under excitation with light. Their approximation is valid as long as light intensities are low enough to avoid any interference of secondary photochemistry with other intermediates [ 14 ]. These intelligent materials also give responses to continuous illumination at high light intensity. There- fore it is important to study the kinetics and equilibria of proton association and dissociation of BR in PM fragments under these conditions. Previously, our research group has concentrated on the behaviour of BR in membrane environment under continuous illu- mination for understanding and modelling of light induced active transport phenomena at physological conditions [ 15-18 ]. The present work has been carried out as an effort to understand the mechanism of action of BR in PM frag- ments at extreme conditions. For this purpose the kinetic analyses of the proton dissociation and associ- ation reactions have been made at different values of pH, temperatures and ionic strength under continuous high intensity light illumination. 2. Materials and methods 2.1. Materials The common reagents used were all of reagent qual- ity and purchased from Merck (Germany). DNAase was obtained from Sigma Chemical Company (USA) and Bacteriological peptone oxoid from BDH (UK). Halobacterium halobium S-9 strain, srcinally obtained from Dr. Khorana's laboratory (MIT, USA) Was a gift of Dr. Mehmet ~im~ek. 2.2. Growth of Halobacterium halobium Halobacterium halobium cells (strain S-9) were grown (as described by Oesterhelt and Stoeckenius [2] ) at 39°C in a sterilized medium containing 250 g NaC1, 20.0 g MgSO4.2HzO, 3.64 g trisodium citrate 5.5 HzO, 2.0 g of KCI and 10 g of bacteriological peptone L-37 in 1000 ml of distilled water at pH 7.0 in a shaking incubator. Growth was followed either by measuring the extinction of the cells at 660 nm or the increase in absorbance of the PM fragments at 560 nm. The cells were left to grow for 7 days. 2.3. Preparation of purple membrane fragments After 7 days, the cells were collected by centrifuga- tion at 6000 rpm for 20 min in a Sorval centrifuge (GSA rotor). The cell pellets were suspended in 20 ml of basal salt solution (the growth medium without pep- tone) overnight at 20°C. Then 0.15 ml of DNAase (2000 units/ml) was added and the solution was dia- lyzed at 4°C against 2 1 of 0.1 M NaCI for 3 days. The lysate was centrifuged at 4500 rpm (2500 g in SS-34) at 4°C for 20 min. The sediment containing small pellets was discarded. The colored supernatant was centrifuged at 19 000 rpm (43 000 g in SS-34) for 60 min at 4°C. The purple pellets were collected and then suspended in 0.1 M NaCI. The centrifugation step was repeated once more. The pellets, recollected and suspended in 5 ml of distilled H20 were stored at - 20°C until used. 2.4. Characterization of the purple membrane The characterization of BR in the above mentioned suspension was achieved spectrophotometrically at 570 nm using an extinction coefficient of 63 000 M- cm-1. The concentration of BR was found to be 25 /zM. The apparent molecular weight of BR determined by SDS polyacrylamide gel electrophoresis as described by Laemmli [ 19], was found to be 26 000 Da 2.5. Measurement of photoactivity of BR in PM fragments PM fragments were suspended in 1 M KC1 to a final concentration of 5 mM. The pH of the suspension was adjusted by adding either 10 mM KOH or HCI.  M. Yiicel et al. /Journal of Membrane Science 104 (1995) 65-72 67 3. Results 1 9.30 8.86 8.42 7.98 ~ ON pH ° ~OFF 9.10 7.54 ~=,,,= = 8. tO 7 55 7.23 7.10 7.05 6.87 6.71 6.66 6.56 6.22 5.78 5.34 4.90 4.46 4.02 3.58 3.14 2.70 0 I .=4~,.~ 6.27 5.90 5.58 5,8 4.62 4.09 5.58 o-~ ~ .n 3.17 I I I I 16 32 48 64 80 Time rein.) Fig. 1. Effect of initial pH on photoresponse of BR in PM fragments at 25°C in 1 M KCI. The photoactivity of BR was measured as pH vs. time by using a combined pH electrode (Russel) con- nected to a NEL pH meter (Model 821, accuracy 0.01 pH, time constant 5 s). The continuous illumination was achieved by a projector lamp (Reflecta) giving a light intensity of 20130 W/m 2 at a distance of 30 cm. The experimental setup was shown in our previous study [ 20], The experiments were repeated at least five times and reproducible results were obtained. 3.1. Effects of initial pH and temperature on photoresponse of BR The photoactivity of BR, in suspension of PM frag- ments was measured at different external pH values (pH 3.09-9.20) at 25°C and the changes in pH versus time were plotted (Fig. 1). It was observed that the light induced proton dissociation activity of BR in the basic pH range is greater than that in the acidic range. However in the dark, the reuptake of protons is rather slow (pH increases slightly). At physiological pH val- ues (pH 6.7 and 7.2), upon illumination, the pH i i° ° ' 20 32 44 56 68 80 Time (rain.) Fig. 2. Effect of temperature on photoresponse of BR, in 1 M KC1 at physicological pH: 10°C (@), 15°C (O), 20°C (×), 25°C (A), and 30°C (F1). 5.80 5.40 /-I t oF, . - i z 5.00 / ~ .,.o-.' 4.20 ~ . ~ 3.80 J I t t 0 16 32 48 64 80 Time rain.) Fig. 3. Effect of temperature on photoresponse of BE at acidic pH: 10°C (O), 15°C (O), 20°C (I), and 25°C ( × ).  68 M. Y~icel et aL / Journal of Membrane Science 104 (1995) 65-72 oN I O 7.00 6.92 6.84 6.76 6.68 6.60 i i i i 20 32 44 56 68 80 Time (rain.) Fig. 4. Effect of KCI concentration on the photoresponse of BR at 25°C at physicological pH. KCI concentration: 0.0 M (V1), 0.1 M (A), 0.2 M (O), 0.5 M (O), 1.0 M ( × ), and 2.0 M ( A ). decreases and returns to its initial values during the dark period. The same type of behavior has been observed in the acidic range but the light induced ApH value is rela- tively small. It should be noted that at low external pH the observed dpH values are very small. Similar pH vs. time profiles were observed also at 20°C. The effect of temperature on the photoresponse of BR was checked at two different external pH values (pH 4.1 and 6.8). Fig. 2 illustrates the effect of temperature on the photoresponse curve of BR at 10, 15, 20, 25, and 30°C at the physiological pH. The behavior of BR has been found to be the same in acidification of the medium upon illumination (proton release) and alkal- inization (proton association) in the dark period. At higher temperatures T > 37°C continuous increase in pH values were observed both in the light and in the dark phases. The effect of temperature on the photo- response of BR in acidic pH region (pH 4.1) is given in Fig. 3. When PM fragments were incubated at an initial pH of 4.0 at 10 or 15°C the behavior of BR changed completely. Upon illumination alkalinization of the medium was observed due to hyper proton uptake. During the dark period, protons were released to the medium (acidification). However at pH 4.0 and at 20 or 25°C, the behavior of BR was found to be similar to the behavior observed at the physiological pH values. 3.2. Effect of different salt concentrations on the photoresponse of BR The effect of different KC1 concentration on the pho- toresponse of BR in PM fragments is shown in Fig. 4. The measurements have been carried out at 25°C and at an initial pH value of about 6.85. As seen in Fig. 4 as the KCI concentration increases the photoenergetic activity of BR indicated as maximum pH change between initial and final values ( A pHm~x - pHo - pHf), also increases and reaches a constant value at around 1.0 M KCI concentration. The effect of different CaC12 concentrations on the photoresponse of BR has also been measured at the same pH value (pH 6.85) and at 25°C in the presence of 0.1 M KCI (Fig. 5). It can be seen that an increase in CaCl2 concentration caused an increase in the photoenergetic activity of BR. 3.3. Kinetic analysis of the photoresponse data Effect of temperature The kinetic analysis of the photoresponse data at 25°C and at initial pH values of 6.55-7.25 has already been reported in our previous publications [15-18]. Here, a similar analysis has been carded out at different temperatures for light and dark phases. As seen in Fig. 6 for the light reactions, the first order kinetic model is valid within the temperature range of 20--30°C around 7.00 t 6.92 6.76 6.68 t 6.60 20 i oN O t I i I 32 44 56 68 80 Time (rain.) Fig. 5. Effect of CaCh concentration on the photorcspons~ of BR at 25°C at physicological pH in 0.1 M KCI. CaCI2 concentration: 0.0 mM (*), 0.5 mM (ll), 2.5 mM (r'q), 5.0 mM (O), and 10 mM (0).  M. Yiicel et al. / Journal of Membrane Science 104 1995) 65-72 69 °1 f Oo O .. I _ ~I: ~ '-~ 1.21- ' ZlXo I- & L . 0.8 +=r= O.al- ' 0.4 ' 0.4 0.0 0.0 lv I 4 8 12 16 4 8 12 16 Time (rain.) Time (rain.) Fig. 6. Kinetic analyses of the light and dark reactions of BR at physicological pH at different temperatures in 1 M KCI: 10°C (O), 15°C (©), 20°C ( X ), 25°C ( • ), and 30°C ([:]). c .J -I.6 -2.0 I -2.4 -2.8 3.2 0 kon i 53 3.4 3.5 _J,_l x lOa(deg ') T Fig. 7. An'henius plots for the light (O) and the dark reactions (0) of BR at physicological pH. The linear model is valid in the region indicated between the arrows. the physiological pH in 1 M KCI solution. However, at 10 and 15°C a multiphasic behavior was observed. A similar behavior is also manifested for the dark reac- tions at the indicated temperatures. The Arrhenious plots for the light and dark reactions of BR in PM fragments are given in Fig. 7. The first order kinetic model is valid in the region indicated Table 1 The first order rate constants of BR at different KCI concentrations for the light (ko,) and dark (kon) reactions at physiological pH and temperature (25°C) KCI (M) kon (min -~) k~ff (min -~ ) ApHm~ (on) 0.1 0.26 0,29 0.04 0.2 o. 19 0,31 0.05 0.5 o. 17 0.21 0.05--0.06 1.0 0.15 0.18 0.06 2.0 0.19 0.23 0.06 between the arrows. This corresponds to the tempera- ture range of 20-30°C. It is interesting to note that the rate constant is the same for light on and light off Table 2 The first order rate constants of BR at various CaC12 concentrations in the presence of 0.1 M KCI for the light (ko.) and dark (kofr) reactions at physiological pH and temperature (25°C) CaCI2 (mM) ko. ApH..~ koff ApH~,,~ (rain -~) (on) (rain -~) (off) 0.0 0.26 0.04 0.29 - 0.04 0.5 0.29 0.04 0.28 - 0.06 2.5 0.20 0.06 0.20 - 0.08 5.0 0.13 0.06 0.32 - 0.10 10.0 0.21 0.05 0.17 - 0.08
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