Plasmon resonance spectroscopy: probing molecular interactions within membranes.

Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.

Enhancement studies on algae and isolated chloroplasts. Part I. Variability of photosynthetic enhancement in Chlorella pyrenoidosa.

Studies of the variability of enhancement in Chlorella pyrenoidosa confirm the existence of two types of variability: a very slow diurnal variation linked to the growth cycle and a much more rapid adaptive response to the immediate incident light conditions (State I-State II transitions). Measurements of the wavelength dependencies and relative contributions of these two types of variability suggest that they may be linked. A close examination of the enhancement signals associated with the State I-State II transition reveals that the transitions can take place in any one of three ways: by a change in Photosystem II efficiency alone, by a change in Photosystem I efficiency alone or by a simultaneous change in the efficiencies of both photo systems. Measurements of the rates of transition between State I, State II and the dark adapted state, Dark, suggest that the behaviour of State II and Dark are normally, but not always, identical. The transitions between the three states were found to be first order. For those samples exhibiting the same behaviour in Dark and State II, the rate of the State I-State II transition was found to be independent of the wavelength of Light II, suggesting that the return from State I to State II is essentially a dark process and that the driving force for the adaptive transition is the over-stimulation of Photosystem I. Finally, a model is proposed, involving an antagonistic control of the quantum yields of photochemistry of the two photosystems, that is capable of explaining the links between the two types of variability, their wavelength dependencies and the shapes of the individual enhancement signals.

Enhancement studies on algae and isolated chloroplasts. Part II. Enhancement of oxygen evolution in intact chloroplasts.

Intact isolated chloroplasts were shown to exhibit a characteristic three-phase pattern of development of oxygen evolution activity. The first phase, Phase I, appeared to be an equilibration phase in which the isolated chloroplasts adapted to the conditions on the electrode surface. It was characterised by a rapidly increasing rate of oxygen evolution accompanied by decreasing enhancement signals. The second phase, Phase II, was an intermediate phase in which the rate of oxygen evolution was maximal and no enhancement was observed. In the last phase, Phase III, the rate of oxygen fell again, normal enhancement was still missing, but the samples appeared to undergo slow adaptive changes closely related to the State I-State II changes previously reported for whole cell systems. The concentrations of Mg2+ within the chloroplast were shown to play an important role in the control of the development of both the oxygen evolution and enhancement signals. It was shown how these signals could be explained in terms of a model that was consistent with that developed in Part I of this investigation to account for the variability of enhancement of the alga Chlorella pyrenoidosa.

Transient kinetic and oxidation-reduction studies of spinach ferredoxin:nitrite oxidoreductase.

The oxidation-reduction midpoint potentials for the two prosthetic groups of the chloroplast-located, ferredoxin-dependent nitrite reductase of spinach leaves have been determined by spectroelectrochemical titrations and cyclic voltammetry. The average of the results obtained by the two techniques are Em = -290 mV for the siroheme group and Em = -365 mV for the [4Fe-4S] cluster. The value obtained for the [4Fe-4S] cluster is substantially more positive than values obtained previously in experiments which utilized electron paramagnetic resonance spectroscopy at cryogenic temperatures to monitor the reduction state of the cluster. Laser flash photolysis experiments have been used to monitor electron transfer from reduced ferredoxin to nitrite reductase and have provided the first evidence for electron transfer between the two prosthetic groups of the enzyme. The effect of ionic strength on the observed kinetics has provided support for the proposal that electrostatic interactions between ferredoxin and nitrite reductase play an important role in the reaction mechanism.

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy.

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0-15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase.

Oxidation-reduction midpoint potentials have been determined, using cyclic voltammetry, for the active-site disulfide/dithiol couples of spinach thioredoxins f and m and of spinach ferredoxin:thioredoxin reductase (FTR) and for a component likely to be the [4Fe-4S] cluster of FTR. Values for the midpoint potentials (n = 2) of -210 +/- 10 mV were determined for both thioredoxins f and m. Two redox centers were detected in FTR, with midpoint potential values of -230 +/- 10 mV (n = 2) and +340 +/- 30 mV, respectively. Alkylation of the active-site cysteines of FTR by treatment of the enzyme with N-ethylmaleimide (NEM) eliminates the component with the -230 mV midpoint potential, allowing one to assign this value to the active site disulfide/dithiol couple. Inasmuch as the only other electron-carrying center known to be present in FTR is the [4Fe-4S] cluster, it appears likely that the high-potential component can be attributed to this redox moiety. The midpoint potential value of the high-potential feature shifts slightly, to +380 +/- 20 mV, in the NEM-treated enzyme.

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase.

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

Presynaptic autoinhibition during rest and sodium-pump inhibition in isolated rat portal vein preparation.

In the presence of cocaine and corticosterone low-frequency (2 Hz) nerve stimulation evoked release of [3H]noradrenaline measured from isolated rat portal vein preparation. In normal Krebs solution exogenously applied l-noradrenaline (3 X 10(-8)-10(-6) M) significantly reduced the nerve-evoked [3H]noradrenaline release. The IC50 value of L-noradrenaline proved to be 1.8 X 10(-7) M. Yohimbine (3 X 10(-7) M) maximally blocked the alpha 2-adrenoceptors and enhanced nerve-evoked [3H]noradrenaline release. In the presence of 5.9 mM external K+, ouabain up to 10(-4) M did not affect either the resting or the stimulation-evoked release of radioactivity from tissues. In the absence of external K+ both the resting and the nerve-evoked release of [3H]noradrenaline increased markedly. When K+ was readmitted to preparations which had been kept in K+-free solution both the resting and the stimulation-evoked [3H]noradrenaline release were greatly reduced temporarily. In K+-free solution L-noradrenaline (10(-6) M) and yohimbine (3 X 10(-7) M) failed to significantly alter the nerve-evoked release. However, 3 X 10(-6) M yohimbine in K+-free solution significantly increased the stimulation-evoked release of [3H]noradrenaline. It is concluded that presynaptic alpha 2-adrenoceptor-mediated "negative feed-back" is present in rat portal vein preparations which can be inhibited by the preferential alpha 2-adrenoceptor blocker, yohimbine. However, if the Na+-pump is inhibited (which by itself enhanced the transmitter release), presynaptic autoinhibition is more pronounced, since a high concentration of yohimbine is required to block it.

Lipid bilayer-based sensors and biomolecular electronics.

The lipid bilayer postulated as the basic structural matrix of biological membranes is widely accepted. Experiments in the early 1960s have made direct studies of lipid bilayers possible. At present, the planar bilayer lipid membrane (BLM) together with spherical lipid bilayers (liposomes), upon suitable modification, serves as a most appropriate model for biological membranes. In recent years, advances in microelectronics and interest in ultrathin organic films, including BLMs, have resulted in a unique fusion of ideas toward the development of biosensors and transducers. Furthermore, recent trends in interdisciplinary studies in chemistry, electronics, and biology have led to a new field of research: biomolecular electronics. This exciting new field of scientific-technological endeavor is part of a more general approach toward the development of a new, postsemiconductor electronic technology, namely, molecular electronics with a long-term goal of molecular computers. Experimental BLMs have been mainly used in the past as models of biological membranes. The methods of BLM studies may not be familiar to those outside biomedical research. Therefore, a brief description of the experimental techniques will be given in Section IV. Recently, it has been demonstrated that BLMs, after suitable modification, can function as electrodes and exhibit nonlinear electronic properties. These and other experimental findings relevant to sensor development and to "biomolecular electronic devices" (BED) will be covered in Section V, after a brief description of biomembranes which have been suggested as nature's molecular devices (Section III). In the last section, the potential use of the BLM system together with its modifications in the development of a new class of organic diodes, switches, biosensors, electrochemical photocells, and biofuel cells will be presented (Section VI). Additionally, this paper, besides presenting a review of our work and those of others on BLMs and liposomes in relation to biosensors and molecular electronics, reports a novel technique for obtaining BLMs (or lipid bilayers) on solid supports. The presence of solid support on one side of the BLM greatly enhances its mechanical stability, while retaining the dynamic properties of the lipid bilayer. Advantages of the new technique for self-assembling amphiphilic molecules on rigid substrates are discussed in terms of their possible uses. That is, the new BLM system (s-BLMs) is potentially useful for technological applications in the area of biosensors, enzyme electrodes, and molecular electronics as well as biochips (Section IV.C). The dividing line between the present microscopic and the future molecular electronics is 1 micron.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of chlorophyll a on intermolecular interactions in liquid crystal.

Studies of the variability of the spectral properties (linear dichroism, polarized fluorescence and the energetic separation of the absorption and emission bands) of chlorophyll a in a nematic liquid crystal matrix with respect to the effects of solute concentration and the external electric fields were made. A close examination of the above mentioned types of variability suggest that the pigment molecules can influence a high initial order of the liquid crystal matrix. These structural changes of the matrix can be explained by the assumption that two types of chlorophyll with different orientations occur: the surface layers with the high degree of orientation, and a central volume of the sample with disordered molecules. The ordered molecules are sensitive to an reorientation by the external electric field.

Deposition of a photosensitive complex within a lecithin bilayer lipid membrane.

In this paper the phenomenon of a photosensitive ion complex of Brilliant Yellow and ferric ions formation in the electrolyte phase and its subsequent deposition within a bilayer lipid membrane (BLM) is described. Deposition of light sensitive complex into the BLM considerably increases its mechanical stability and drastically changes its electrochemical and photoelectrical properties as well.

Solid state investigation of mefloquine hydrochloride.

Mefloquine hydrochloride was crystallized under different conditions and the products were studied by thermal analysis, IR spectroscopy and X-ray diffraction. It was demonstrated that different conditions of crystallization resulted in different crystal structures. The alpha- and delta-modifications were hydrates, the beta- and gamma-forms were polymorphs, the epsilon-form was an acetone solvate and theta-mefloquine was tetrahydrofuran solvate. During long storage at room temperature, the alpha-, delta- and theta-forms of mefloquine hydrochloride were transformed. By heat treatment of the delta-, epsilon and theta-modifications, a new crystal structure was obtained.

A self-assembled pigmented BLM on a platinum support: the light-induced electrical effects.

The light-induced voltage and current changes under continuous illumination have been investigated in pigmented self-assembled lipid bilayer membranes deposited on a platinum electrode. Such self-organized pigmented bilayer-platinum system containing Zn-Phthalocyanine (ZnPc) as a photosensitizer and glycerol-dioleate (GDO) as a bilayer forming solution has been found to shift its electrode potential to more positive value on light irradiation as well as to increase the cathodic current across the membrane. The results indicate a direct electron transfer from the platinum electrode to hydrogen ion in the electrolyte solution. Furthermore, it has also been demonstrated a dramatic increase of the photocurrent over the time course of BLM formation visualizing a role of the bulk quenching processes which are significantly diminished in thin bilayer membrane.