Membrane potential is involved in regulation of photosynthetic reactions in the marine diatom Thalassiosira weissflogii

High-intensity Chl fluorescence transients (OJIP transients) and light-induced kinetics of the delayed light emission were measured in diatom microalga Thalassiosira weissflogii in the presence of various uncouplers and photosynthetic inhibitors. The I step in the OJIP transients in T. weissflogii was essentially reduced or completely absent but was restored in the presence of uncouplers valinomycin, FCCP, and nigericin. Moreover, valinomycin enhanced ΔpH-dependent non-photochemical fluorescence quenching following the OJIP rise. In the presence of valinomycin, the transthylakoid membrane potential was significantly inhibited as evaluated by measurements of the delayed light emission. The results suggest a membrane potential control of the fluorescence yield in T. weissflogii. Possible mechanisms underlying the observed effects of uncouplers are discussed.     

ALLOPHYCOCYANIN FORMS ISOLATED FROM NOSTOC SP. PHYCOBILISOMES*

Abstract— Allophycocyanin from dissociated phycobilisomes of Nostoc sp. occurs in three spectrally identifiable forms that fractionate on calcium phosphate adsorption chromatography as: allophycocyanin (APC) I (15–20%), APC II (4&50%), and APC III (30–40%). APC I has a single absorption maximum at 654 nm, and a fluorescence emission peak at 678 nm. The absorption peaks of APC II and III are both at 650 nm, but the relative absorbance at 620/650 nm of APC III is less than that of APC II. The emission of both is maximum at 660 nm. On zone sedimentation in sucrose, their S_20,w values of 6.0 ± 0.1 (APC I), 5.0 ± 0.1 (APC II), and 5.3 ± 0.2 (APC III) were comparable to the order of their elution from Sephadex G-200. On SDS acrylamide gel electrophoresis two subunits were resolved with apparent molecular weights of 16,900 and 18,400 daltons. When stained by Coomassie blue, they were present in a ratio of 1α:1β in APC II and III, and a probable ratio of 2a:3β in APC I. The larger size of APC I may be accounted for by additional β subunits, by the presence of an additional polypeptide of 35,000 daltons, or both. Over several days, bleaching as noted by a decrease in absorbance at 650 nm, occurred in all three forms; in addition, the more pronounced bleaching at 650 nm, relative to 620 nm, results in APC III becoming spectrally identical to APC II. A trace of a fourth pigment, probably comparable to allophycocyanin-B, was occasionally detected. The results suggest that several in vitro APC forms (sharing similar subunits) arise upon phycobilisome dissociation, and that APC I is the form most closely related to the final fluorescence emitter of intact phycobilisomes. In this form it probably serves as the bridging pigment in energy transfer from the phycobilisomes to chlorophyll.     

Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements.

Understanding the role of oxygen in the physiology, pathophysiology and radio- and chemosensitivity of animal cells requires accurate and non-invasive measurements of oxygen concentrations in the range of 0-2x10(-4) M, in cells in vitro or in vivo. High resolution 3D imaging techniques could be particularly useful in investigating tissue oxygenation in vivo and in model tissues (multicellular spheroids) in vitro. The goals of this work were to develop microscopy techniques and (i) to define conditions under which two oxygen-sensitive luminescent dyes, Ru(bipy)(3)(2+) (tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate) and Ru(phen)(3)(2+) (tris(1,10-phenanthroline)ruthenium(II) chloride hydrate) can be used to probe oxygen concentrations within viable cells in vitro, when no phototoxic effects are evident, and (ii) to investigate the mechanism of phototoxicity once cell damage occurs. This report demonstrates that Ru(bipy)(3)(2+) and Ru(phen)(3)(2+) do not pass through intact biological membranes, do not cause measurable photodamage to plasma membranes at a concentration of 0.2 mM and, when loaded into endosomes, yield a strong luminescent signal. However, at an extracellular concentration of 1 mM, in the presence of 457-nm light, detectable amounts of both complexes accumulate at the plasma membrane and cause a loss of membrane integrity via a mechanism which may involve the generation of singlet oxygen.     

EXCIPLEX BY PYRENE-N,N-DIMETHYLANILINE PAIR BONDED TO POLYMERS IN AQUEOUS MEDIA*

Abstract— Emission spectra of poly[2-(4-N,N-dimethylaminobenzyl)-2-(l-pyrenylmethyl)ethyl methacryl-ate] (I) and the copolymers with vinyl-benzyltriethylammonium chloride (II) or sodium p-styrenesulfo-nate (III) were studied in extremely polar media such as dimethylformamide (DMF) and water. While the emission by the monomer model compound (2-(4-N,N-dimethylaminobenzyl)-2-(l-pyrenylmethyl)-ethyl isobutyrate(IV)) scarcely showed exciplex emission in DMF or aqueous DMF, exciplex by I was clearly observed in the same solvents. Furthermore, the ratio of exciplex intensity (F_e) to monomer intensity (F_m) increased by the addition of water to the DMF solution up to 60 vol%. This abnormal spectral behavior of increasing exciplex emission intensity with solvent polarity was interpreted as being due to shrinking of polymer chain. The peaking wavelength of exciplex shifted towards blue in the presence of water, indicating that the solvation of exciplex was hindered and/or the hydrophobic domain was organized. This interpretation was supported by the exciplex emission of II in water. No exciplex was detected from III in water. This is the first example of exciplex emission in homogeneous aqueous solution. In comparison with the published results of micellar systems, the exciplex emission of the polycation indicated that the hydrophobic domain in the polycation was so strong that the solvation of exciplex was considerably hindered. The peaking wavelength of exciplex at 480 nm also lends support to the presence of a non-polar microenvironment.     

Enantiospecific assay for mammalian carbonyl reductase by liquid chromatography with fluorescence detection

A fluorogenic substrate with an unsymmetrical carbonyl for the sensitive assay of mammalian carbonyl reductase activities, 4-(6-methoxy-2-benzoxazolyl)acetophenone (I), has been prepared. The fluorescence quantum yield of I in acetonitrile is 0.12 at the emission maximum of 448 nm. The corresponding racemic alcohol produced by the chemical reduction of I, (+/-)-sec.-[4-(6-methoxy-2-benzoxazolyl)]phenethyl alcohol (II), exhibits ca. three- to fourteen-fold higher fluorescence at a shorter wavelength emission maximum of 370 nm in conventional solvents. Each enantiomer of II is sufficiently resolved on a chiral cellulose high-performance liquid chromatographic column without derivatization and quantified with high reproducibility. The detection limit for II is 20 fmol per injection at a signal-to-noise ratio of 3. The validity and applicability of I are evaluated with cytosols of mammalian tissues. The optimal pH for metabolic reduction of I in rabbit liver cytosol preparations is 6.2 in the presence of NADPH. The metabolism is proved to be highly stereoselective. The resulting alcohol produced by mammalian tissue preparations, except rabbit kidney, is predominantly of the S-(-)-configuration.     

THE COMPLEXITY OF THE FLUORESCENCE OF CHLORELLA PYRENOIDOSA

Abstract— The complexity of the room-temperature emission spectrum of Chlorella was investigated by a matrix analysis method. This approach revealed the presence of two independently fluorescent components in the short-wave region of the spectrum. These components, maximal at about 687 and 695 nm, appeared to correspond to the fluorescence of the bulk pigments of PS II and PS I respectively. The analysis was insensitive to the individual species within the photosystems. As such, other minor fluorescent species, usually observed at low temperatures, which presumably correspond to fluorescence from the trapping centres, did not appreciably complicate the analysis. The absorption spectra of the two photosystems were calculated from the fluorescence data. The results were similar to those that have been obtained by other workers from oxygen evolution and DCMU poisoning data but differed from those obtained by computer analysis of the absorption spectrum. Addition of reduced DCPIP was observed to reverse the increase in fluorescence yield and changes in the spectral distribution of emission taking place on poisoning the algae. The correlation between this and the catalysis of photophos-phorylation in aged or poisoned chloroplasts was noted. This correlation was tentatively interpreted as evidence for a direct interaction between the donor system and the photochemical apparatus associated with PS II, rather than with a member of the electron transport chain as is normally assumed.     

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

An AIE probe for long-term plasma membrane imaging and membrane-targeted photodynamic therapy

Two red-emissive luminogens (TPTH and TPTB) with typical aggregation-induced emission characteristics were developed. By introducing the heavy atom of Br at the end of alkyl chain, TPTB exhibited higher reactive oxygen species generation efficiency through both types I and II pathways. Due to its excellent biocompatibility and proper lipophilicity, TPTB could be used for long-term cell membrane staining and this staining ability was independent of the change of plasma membrane potential. Furthermore, TPTB could ablate the cancer cells through cell membrane-targeted photodynamic therapy.     

CORRELATION BETWEEN DELAYED LIGHT EMISSION AND FLUORESCENCE OF CHLOROPHYLL a IN SYSTEM II PARTICLES DERIVED FROM SPINACH CHLOROPLASTS

Abstract— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn²⁺, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X^-Y⁺) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X^-Y⁺) into (X^-Y). Since the form (X^-Y⁺) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X^-Y⁺) into (X^-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.     

一种新的DNA探针-2-(N-二茂铁酰氨丙氧基)-6-氯-9-氨基吖啶的合成与性质

本文合成了一种含二茂铁侧基的DNA荧光探针-2-(N-二茂铁酰氨丙氧基)-6-氯-9-氨基吖啶, 并对该化合物的紫外可见光谱, 荧光光谱, NMR谱以及它和小牛胸腺DNA的作用与二茂铁侧基的吖啶作了比较和讨论。     

Effect of the presence of iron vapors on the volumetric emission of Ar/Fe and Ar/Fe/HZ plasmas

The net volumetric emission coefficient was calculated using the escape factor method for Ar/Fe and Ar/H₂/Fe plasmas, at atmospheric pressure, over the temperature range from 3000 K to 30,000 K. The calculation involved 712 lines for Ar I, Ar II, and Ar III, 3481 lines for Fe I, Fe II, and Fe III, and 230 lines for H in the Ar/H₂/Fe case. A semiempirical method was used for the determination of line profiles and line broadening. The results show a strong influence of the presence of even traces of iron vapors at low temperatures where the volumetric emission increases by several orders of magnitude. Special attention is given to self-absorption of the argon resonance lines which prevents the radiation from escaping within a few millimeters from the emission source.     

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77K emission spectra revealed that Mg2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.     

CdS量子点的制备及细胞膜初步荧光标记

以巯基乙酸为稳定剂,通过控制反应温度、反应时间及pH值,在水相中合成了稳定的受激发出紫光、蓝光、绿光、黄光和红光的CdS量子点;通过紫外可见吸收光谱、荧光光谱和X射线衍射谱(XRD)对产物的光学性能和晶体结构进行了表征,结果表明所合成的CdS量子点分散性较好,量子产率为8%,为立方晶型,粒径约1 nm;利用荧光倒置显微镜观察了量子点在洋葱内表皮细胞膜上聚集及受激发射荧光行为,实现细胞膜初步标记.     

Cytochrome b6/f complex as an indigenous photodynamic generator of singlet oxygen in thylakoid membranes

Possible association of photodynamic sensitization by cytochrome b6/f complex (cyt b6/f) via singlet oxygen (1O2) mechanism with photoinhibition damage to photosystem II (PS II) was studied using such subthylakoid preparations as photosystem I (PS I) particles, PS II core complex and cyt b6/f from spinach leaves. Upon exposure to bright light, PS II core complex lost photosynthetic electron transport activity to a certain extent, whose-spectral dependence implied that pheophytin a is likely involved in photoinactivation of PS II core complex in itself. The presence of PS I particles exerted virtually no effect on PS II core photoinactivation. However, the inclusion of cyt b6/f in samples resulted in a marked exacerbation of the photoinactivation, particularly in UV-A and blue light. Such effect of cyt b6/f was suppressed by azide and enhanced by the medium deuteration. Photogeneration of 1O2 from cyt b6/f was confirmed by ESR and spectrophotometry, chemically trapping 1O2. Action spectra for both 1O2 photoproduction and PS II core photoinactivation by cyt b6/f bore a close resemblance to each other, seemingly carrying the absorption characteristics of the Rieske Fe-S protein. A complex deficient in the Rieske protein prepared from intact cyt b6/f showed virtually no generation of 1O2 in light, whereas an efficient photoformation of 1O2 was seen in the Rieske protein preparation. The results suggest that cyt b6/f, rather specifically the Rieske center, may play a prominent role in photoinhibition processes through type II photosensitization in thylakoids.     

Kiwifruit Actinidin: A Proper New Collagenase for Isolation of Cells from Different Tissues

Actinidin is a cysteine protease abundant in Kiwifruit. This enzyme is known as a meat-tenderizing protease. In this project, actinidin was purified from kiwifruit by salt precipitation and ion exchange chromatography. Collagenolytic effect of the purified enzyme was tested in four different buffer systems. Thereafter, the enzyme was used for isolation and culture of cells from three different tissues: endothelial cells from human umbilical vein, hepatocytes from rat liver, and thymic epithelial cells from rat thymus. Our results revealed that actinidin can hydrolyze collagen types I and II at neutral and alkaline buffers. Furthermore, actinidin compared with type II or IV collagenase isolated intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90%. These results address a novel and valuable collagenase, which can be used efficiently for hydrolysis of collagen and isolation of different cell populations from various solid tissues.     

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.     

CINETIQUE DES ECHANGES D'OXYGENE ET DE LA FLUORESCENCE DES CHLOROPLASTES ISOLES

Abstract Oxygen evolution and fluorescence have been studied with isolated chloroplasts illuminated, in the absence of Hill reagents, by flashes or continuous light. As in whole cells, at least two substances are involved in the primary process leading to the oxygen evolution. The first, called E, probably is the photochemical “complex” of System II. After a long period of darkness, E is not active. It is activated in two steps. Step one is a photochemical reaction, induced by a quantum of light absorbed by pigment-system II, which results in the production of E in a reduced state. Step two is a dark oxidation of the reduced E by the second substance, A. The oxidized E can then enter the normal photochemical cycle of system II. Reduced E might alternatively be oxidized by oxygen, this reaction being responsible for a very rapid and brief light-induced oxygen uptake. Substance A is measured by the oxygen burst and is present in the chloroplasts at the approximate ratio of 1 molecule for 70 molecules of total chlorophyll while E is at the ratio of about 1/2800. This gives a E over A value of 1/35 which is much smaller than the one found in whoe cells (ca. 1/10). This independent behavior of E and A suggests that chloroplast extraction destroys some photochemical centers without having a direct impact on A, which might diffuse from one center to another. Besides the brief light-induced oxygen uptake above mentioned, there is another one which is related to System I functioning. The kinetics of the oxygen evolution and of the fluorescence have been compared. During the activation process of the oxygen evolving ability, rate of oxygen evolution and fluorescence yield increase in a parallel way. After the maximum velocity of the oxygen burst is reached (i.e. after activation), the fluorescence yield keeps growing up until the steady-state is attained (with an intermediary plateau), whereas the rate of oxygen emission slows down. The time-course curves of fluorescence obtained with inactivated or activated chloroplasts are essentially different in that the initial yield is higher in the latter case.     

PHOTOSYSTEM II HETEROGENEITY IN THE MARINE DIATOM Phaeodactylum tricornutum

Abstract— The kinetics of photosystem II photochemistry are analyzed in the marine diatom Phaeodacfylum tricornutum by measurement of fluorescence induction in cell suspensions treated with 3–(3,4-dichlorophenyl)-1,1-dimethylurea. Photosystem II kinetics are found to be biphasic, the sum of two exponential components, suggesting that biphasic energy conversion in photosystem II may be a general consequence of thylakoid membrane appression. The emission wavelength-dependence of fluorescence induction suggests that the two photosystem II components have different variable fluorescence emission spectra. The slower component exhibits characteristic emission of the diatom light-harvesting complexes while emission from the faster component resembles that of the photosystem II reaction center. Variable fluorescence emission (293 K) at wavelengths > 700 nm is assigned to photosystem II. Application of model equations indicates that the two photosystem II unit types differ primarily in antenna size. A new analytical procedure is presented which eliminates ambiguities in the kinetic analysis associated with the incorrect assignment of the maximal fluorescence yield.     

Effect of Membrane Potential on the Binding of Merocyanine 540 to Human Erythrocytes

Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca₂+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.