Photoelectric Effects in Lipid Bilayer Membranes A Pedagogical Review Jay S. Huebner, Arm E. Popp,' and Kenneth R. Williams Center for Membrane Physics and Department of Natural Sciences, University of North Florida, Jacksonville, FI. 32216
Charge rearrangements in and betumeen molecules are of fundamental importance in chemiral reactions. Vet chemistry majors are not normally exposed to experiments in which charge displacements or rearrangements can be observed. The reason for this has uossiblv been the lack of awuronriate is to apparatus and procedures. he purpose of this demonstrate that this situation has chaneed. Photoelectric effects in hilayer membranes provide convenient experiments in which charge displacements occurring over molecular dimensions can he observed in laboratory exercises that are appropriate for undergraduate chemistry and biochemistry majors and beginning graduate life sciences students. This paper (1) provides information appropriate for introductory lectures on photoelectric effects in membranes, (2) describes the apparatus and supplies required for laboratory exercises. (3) outlines tvuical laboratorv exercises. and (4) identifies the chromoph;;res known to iiduce photoelectiic effects. some for which the mechanisms are unknown. Experiments with these chromophores, and with other untried chromonhores. can arovide useful subiects for undereraduate student researcl;. Photoelectric effects in bilaver . lipid - membranes were initially studied by Tien (1,2), who reviewed the early literature (3). The initial impetus for these studies came from their relevance to biological processes. The connection to photosynthesis and visual transduction led to the study of photoelectric effects from chlorophyll and rhodopsin, identified below. Additionally, it has been argued that these effects are useful for studying the membrane transport processes integral to nerve impulse conduction and respiration
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A modest apparatus consisting of a stroboscope and storaee oscilloscone, described below. enables charee disnlaceGent time constants ranging from a few micr&ecoxids to several seconds to be recorded accuratelv bv undergraduate students with no prior experience with s;chapparak. Students having experience with oscilloscopes in other courses (6),or those taking undergraduate research, learn to operate a pulsed-laser, 60-MHzoscilloscope system that has 10-ns resolution. This approaches,the high sbeed limit for recording signals from a single planar membrane, the limit resulting from the limited conductance of aqueous solutions (7,8, also see appendix). Using suspensions of memhrane fragments, photosynthetic organelles, and lipid vesicles, however, Trissl et al. (9-11) have extended the charge displacement resolution into the 100-ps range. Although apparatus required for such resolution currently seems too expensive for undergraduate laboratories, it is clear that the future holds exciting possibilities for applying the results of this ongoing research to chemical education. Photoelectric effects in membranes are defined as transmemhrane voltage or current fluctuations resulting from illumination. Brief light flashes are typically used in the
' Present address: Georgia institute of Technology. Atlanta. GA
30332.
it may be helpful for readers to know that some controversy surrounds interpretations of some photoelectric effects (3-5).
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Journal of Chemical Education
I
LIGHT FIBER
PANEL
Fioure 1. A diaaram of the exoerimental arranoement used to obtain ohotae eclrlc effeclsfrom ollayer membranes Scnemalfcd agrams of tns contro Pawl i ght tr gger and electrometer c rc.lts and other conrtr.cllan detads have oeen p ~ b l l r h e delsewnere (7)
ground -
meter
-D-
12
3
Figure 2. A simple parallel-plate capacitor model of a bilayer membrane, in which electric charger displaced within the dielectricalter the transmembrane voltage. mese voltage variations are detected by an elecbometer. The pasitive charge movements illustrated here, from point #Ito 2, and either on to 3 or back to 1, produce the voltage variations illustrated, which are also dis~ u s s e din the text.
arrangement illustrated in Figure 1. Apparatusronstrurtion and other experimental derails are provided elsewhere (12, 71. The control circuit initiates an oscillosco~esn,eeo. durine which the light is flashed. Voltage transients b; the memhrane are displaved on the oscillosco~ealone with a photocell trace, which in some arrangements can Gve the time, duration, and intensitv of the lieht flash. The traces the oscilloscope screen, may be recorded by or by using one of several commercial digital oscilloscopes now available, some of which can also he interfaced to microor minicomputers. Simple control experiments are used to verify the signal's origin and that any artifacts have been properly limited. These experiments include flashing the light (1)prior to adding the chromophore, (2) after the chromophore has been added but with a card to block the light so it does not reach the membrane. and (3) after the memhrane has been ruptured. The simplest physical model of a bilayer membrane is a parallel plate capacitor in which the aqueous solutions form
conductor plates and the lipid hilayer forms the dielectric. In the usual arrangement, the outside solution connects to electrical ground while the inside solution connects to the active (positive) electrometer input. The movement of electric charges across the capacitor dielectric result in transmembrane voltage variations, as illustrated by the following example. If positive charges leave the outside membrane surface ( # 1 in the Fig. 2) and move with constant velocity across the dielectric, then a linear voltage ramp will he generated, as illustrated on the right, reaching a peak voltage when the charges reach position #3. The memhrane voltage is given by V ( t )= nqd(t)lCD
(1)
where n is the number of charges q that have moved a distance d(t) across a membrane of capacitance C and thickness D. On the other hand, if the positive charge in Figure 2 stops moving at position #2, and then after a time moves back to # 1, the membrane voltage will stop rising, and then
Table 1.
Suggested Supplles and Equlpment for Recording Photoelectric Elfects from Bllayer Membranes
Acid dichromate cleaning solution
Gommercialiy available ~ h r o m o p h w e s d i s ~ ~ ~inStext Bd Acid Red 88 chlorophyll s o r b d106~3
Tvptophan Electrodes. AglAgCilblack cloth
Am. Scientific Pmducts. Ocaia. FL. for cleaning the Pyrex and Tefion membrane ceiis (which should n d be cleaned with detergenh).
Aldrich Chem. Co., Milwaukee. Wi Sigma Chemical Go.. St. Louis. MO Nippon Kankoh. Okayama. Japan (other cyanine dyes are available from Eastman Kadak Co.) Sigma Chemical Co. custom made by wrapping black cotton cloth m u n d plaled Ag wire (7) made by filling disposable pipet tips with 5 % agar in eiectroiym solution custom made from operational amplifier (see 7)
Farsday cage
custom made by covering plywwd box with wire screen Pulsed iasersare supplied by: Coherent. Palo Alto, CA; Coop: Laser, Sunnyvale. CA; Lambda Pnysik. Acton. MA; Lumonics. Otlawa. Ontario. Canada; Photo~hemicaiReswarch A ~ s o ~ i a t eOak s . Ridge. TN; Quantei. Santa Clara. CA; and Spectra-Physics. Piscataway. NJ.
glass fushed silica (UV to 260 nm) reflective interface (UV to 200 nm) Lipids choiesteroi glycerol monoolein phosphatidyicholine pho~phatidylethanolamine
fall hack to zero. as illustrated bv the dashed trace. It is assumed in this discussion that charge movements are iniriated bv flash illumination. so the resultina m ~ m b m n evoltage vaiiations are referredto as photovoltages. It is useful to think of bilaver membranes as ordered structures that tend to order absorbed chromophores in space so that charge displacements, synchronized by light flashes, may he detected. The following points summarize important details of the relation between charge displacements and the observed photovoltages. (1) The photovoltage amplitudes are proportional to the numher of charges moving times the distance of their displacement. Since bilayer membranes are essentially two lipid molecules thick, the photovoltages are the result of charge displacements over molecular dimensions. As a rule of thumb, 3 X lo7 elementarv charaes ver mm2 of membrane area crossina a hilaver d i prod&e~voltagetransienr of-1 mV amplitude. r2)'I'he ~hotovolraaewaveforms closely follow the kineticsofchar~e displaceme& within the ~imitsbfthe electrometer and oscil-
Membrane ceiis. external Pyrex (for visible light and near UV) quartz Spectmmeter ceiis Membrane cells, internale Teflon t 0 r m beaker(& = 50 pF) Tefion BmLbeaker (G,= 10 pF) polyethylene beaker (6,= 10 pF)
custom made (see Fig. 3) Fisher Scientific Co.. Pittsburgh. PA
Chemwave. Chempiast. Wayne, NJ custom made, 5-mm-thick wails custom made (see 15)
Micropipet. Oxford Sampler. 100 pL, with disposable tips
Oxford Lab.. Foster City. CA
Microscope, dissecting
Nikon kc.. Garden City. NY. for viewing membranes
Nitrogen "dry" bags, for degassing decane and preparing lipid soiutions
instr. for Research and industry, Cheitenham, PA
Optical filters, for limiting the strobe light to the membrane
h i e l Corp., Stamford, CT
Oscilloscope on screen storage, to 2 MHr
~ o d e5103 i N. ~ektronix.Baaverton. OR digital storage oscilloscopes are available from Gouid Biomation. Santa Clara. CA: LeCroy, Spring Valley. NY: ~ i c o i e i hiadison. . W< and Tektronix. Beaverton. OR
Stirring motor, air or water driven
Lab. Supplies Co.. Hicksviiie. NY
Strobscope
MDdei t539A with P4 capacitor (7). GenRad, Concord. MA
Edmund Scientific. Barrington. NJ EGBG. Salem. MA Oriel Corp.. Stamford, CT
Slrobosco~)flash Nbe. xenon with glass envelope xenon wilh fused silicaenvelope xenon with quartz window
GenRad. Concwd. MA EGBG. Salem. MA EGBG. Salem. MA
added to decane at 10 mg/mL, and stored frozen (-5 'C) in nitrogen. Sigma Chemical Co. Sigma Chemical Go. Sigma Chemicai Co. Sigma Chemicai Co.
Syringe for dispensing membrane lipid, with repeating dispenser
# 7tON, Hamilton Co., Reno. NV # PB 600. Hamilton Ca.
Timing circuit
custom made (see schematic in 7)
Vibrational isolation system
custom made concrete slab suspended on tennis bails, w with air pressure in rubber bladder. Commercial madeis are avaiiabie from Backer-Lorino Goro.. . . Peabodv. MA. and Magfloat, Hopwell Co.. Htdrnn . . . ., NH
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lmarnal Teflw cells wnh unshleldedr l m slecncdes areaim available hom FTR Lab.. Jersey City. NJ
Volume 65 Number 2
February 1988
103
0..fR'] -
TOP VIEW
SIDE VIEW
Figure 3. Detai~sot me Pyrex ode, memorans cell wtn approximate d men. dons me lube aoametsr ( i d ) snould be chosen to prov~dea snLg lit far ma inside membrane cell
loscope slewing rates, t h e flash duration, and RC time constants described below. (3) T h e photovoltage observed when several different mechanisms independently displace charges is the linear sum of the photovoltages resulting from each mechanism separately (13). (4) T h e production andlor reorientation of dipoles shoqld also contribute t o the photovoltaees. althoueh sienals knownto occur bv this mechanism h a v e h o i been ZescXbed (14). A d i s c u s s i o ~of mechanisms which disolace charges in bilaver membranes is orovided below. ~ e k n e m e n t to s the parailel plate capacitor model of bilayer membranes are given in the appendix. Laboratory Exercises The following exercise generally requires two 3-h laboratory periods for uninitiated students when startine with the anoarstus setun. .. and lipid-decane and ehromophore stock solutions prepared. The items needed are listed in Table I. The first lab period is spent with students learning cooperate various partsoftheapparatus, form~ng bilayer membranes and measuring their capacitance and resibranee, steps 1 through 4 behrw. In the second period, durinl: which steps 1 thruurh :are typically completed, those skills are reinforced and used to obtain photov&ages~fromhilayer membranes. Step I . Rinse a clean, assembled membrane cell with the desired aqueous electrolyte solution. After the final rinse, wipe a few drops of lipid-decane solution on the inside and outside of the Teflon cup to "saturate the cell", and fill with aqueous electrolyte solution. Stir for -10 min, while preparing electrodes. Step 2. Place agar-salt bridges in the electrometer assembly, and insert cloth-wrapped AgIAgC1 electrodes, ensuring that the electrodesare immersed to make good electrical contact. The combined electrode resistance, ZR., can be measured at this time by inserting the agar tips in aqueous electrolyte, and measuring the resistance between the silver wires with an ohmmeter. Step 3. Place the filled membrane cell in the Faraday cage, set the light source to law intensity, and turn on the light oscillator switch. Position the membrane cell so the light beam passes through the membrane hole, and adjust the microscope for clear viewing of the hole. Position the electrometer assembly so the agar tips contact the inside and outside aqueous electrolyte solutions. Set the oseilloscope vertical amplifier to 10 mvldiv. and the time base to line trigger and 1sldiv. Step 4. Farm a membrane by wiping the end few millimeters of the syringe needle across the membrane hole, adding lipid-decane solution as required. A reflecting film or membrane should be observed that will thin to form colored bands indicating that the membrane's thickness is on the order of the wavelength of visible light. The membrane resistance and capacitance may now be measured by applying *40 mV through the shunt resistor (7). The capacitance will inerease as the membrane thins, and in a few minutes it should form a "black" or bilayer area that will spread overthe center of the hole. If the film does not thin, then prohahly there is too much lipid-decane solution in the hole. It can he drained by touching the top with the needle tip, causing lipid to flow upward along the needle. Bilayer membranes are black in reflected light, indicating that their thickness is much less than a wavelength of light. The capacitance values will depend on the specific lipid used, but t,wical values are 3 to 9 nF/mmZ(3). Step 5. Add a quantity of the chromophore stock solution to the inside solution by micropipet and stir by "pumping" the micropipet and/or magnetically stirring the solutions.
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Journal of Chemical Education
Figure 4. Photovoltage induced by chlorophyll b, 5 mM KSFe(CN)aand 437-nm Is~erflashesof10-nsduration.Thechloraphyil bldioleoyi phosphatidylcholine (111 by weight) bilayer was prepared in 0.1 M NaCI aqueous solutions. The high-speed trace was recorded by signal averaging two traces on a Tektronix 468 orcllioscope and transferred to an Apple lle microcomputer on an IEEE488 (GPIB) interface. it is time resolved, and shows a risetime of -150 ns. The short bars represent 40-ns voltage samples. The slower traces were recorded on an 5103N osciiioscope and photographed. The dashed trace at 0.5 sldiv. is the membrane's RC time. described in the text.
Figure 5. Photovoltages from 2-mM L-tlyptophanand UV strobe flashes on phosphati~lethanolamincdecanebilayers prepared in 1 m M NaCi aqueous solutions. SUCCBSS~VB traces were stored an a Tektronix 5104N storage oscilloscope at lower sweep speeds, and photographed.
Step 6. Adjust the light to high intensity, and operate the control circuit to trigger the oscilloscope and light flash to observe the photovoltage. Adjust the oscilloscope vertical gain, sweep speed and control circuit trigger delay as required to observe the photovoltage at high, intermediate and low sweep speeds, so as to clearly display the amplitude and all time constants. Photographor record traces as desired. It is also often useful to record the membrane's RC time, and a "noise trace", which results when the light is blocked from the membrane. Steo 7. Alter various conditions. such as the lieht intensitv. ,. snecrral composition or wavelength, and direction of polarization, the dye concentrarion, the inside or outaide solution pH, and/or the electrolyte concentration, e t r . to test ideas eoncernrng the possible mechanism(s)producing the photovoltage.
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Mechanlsms Table 2 lists the chromoohores reported or known t o us t o give photoelectric effects in bilayer membranes. Five are discussed here to illustrate i m w r t a n t details. Four of these are available commercially, wh:lchmake them convenient for teaching exercises. Photoredox Reactions Chlorophyll and other porphyrins transfer electrons t o adjacent oxidizing agents when excited by light (34). Membranes oreoared from lioid solutions containinh chloroohvll ." in symmetrical aqueous solutions are symmetrical, and so will not produce photovoltages unless some asymmetry is introduced. Oxidizing agents have been widely used t o create membrane asnnmetries and therefore ohotovoltaees (27, 35-38). ~ i ~ u 4r illustrates e this type of b h o t o v o l t ~ e , which is described a s being biphasic since it becomes both negative and positive. T h e fast negative voltage excursion is caused by electron displacement. T h e mechanism(s) producing the slower charge displacements evident in the photovoltages remain t o he explained. &
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Photoelectron Ejection Trvutoohan is known to be ohotoionized b v -280-nm light;; a~ueuussolutions(39). ~ i g u r 5e illustrates phutovoltages that result from the 1JV illumination of hilayer mem-
hranes with tryptophan added to the inside solution (15). The initial negative voltage excursion results from absorbed tryptophan ejecting electrons, which move toward the inside aqueous solution. The -7-ps risetime results from the -7-ps duration of the strohosco~eflash. The slower nhotovoltaee components persumably result from mobile tryptophan ur other radicals hut have not heen studied. Additional discussion may he found elsewhere (15).
moving from absorbed dye toward the nqueoussolutions. ( 2 ) Photodeprotonated dve is re~rotonatedfrom the adiarent solutionsat a rate that depends on the solution pH. (3j Acid Red 88 permeates bilayers a t a limited hut sufficient rate to produce smaller signals from the outside membrane surfaces. Therefore the photovoltages observed are the sum of the waveform produced by the inside surface and the inverse
Photoacidification
Many compounds are known that change their DK,'~(i.e., become stronger or weaker acids) upon photoexri;ation (40. 4 1 ) . Under appropriate conditions, some of the compounds that become sironger acids produce photovoltages hyreleasing protons near the memhrane surface (33). These protons move rapidly into the adjacent aqueous solution. Figure 6 illustrates signals produced by Acid Red 88, along with its structure. Traces A and B were ohtained in DH5.5 solutions. The risetime was unresolved (i.e.,
