Ⅰ. INTRODUCTION
Tea and coffee, the most common drinks served nearly every day to the vast majority of people in the world. Tea, more precisely the aqueous extract of tea leaves, had been shown to have antibacterial1, antiviral2 and protein denaturating 3 activities in vitro. These biological activities of tea extracts are attributable to the presence of polyphenol compounds 4, collectively termed catechins. Catechins inhibit the growth of bacterial cells and the bactericidal activity is much higher against Gram-positive than Gram-negative bacteria4.
Mammalian viruses treated in vitro with tea extracts showed reduced infectivity to cultured cells or embryonated egg2. Polyphenols extracted from tea and coffee, collectively termed catechins, showed similar antimicrobial effects 4. Catechins acted on microbes, but also on enzymes5 and hemolytic toxin6, indicating that the compounds deactivate proteins. Catechins also exert their effect on mammalian cells, inducing lymphocyte proliferation, immunoglobulin synthesis7, and mitogenicity of B-lymphocytes8, and stimulating the interleukin production of human leukocytes9 at a low concentration. These biological activities correlate with the presence of galloyl and gallic moieties of the catechin structure. (-)-Epigallocatechin gallate has the strongest biological activity and (-)epicatechin has the least4.
The mode of catechin action was explained by suggesting that catechins exert an effect on the membrane, changing the fluidity10, morphology11, and decreasing the flux of thiourea and cycloleucine12. The immuno-enhancing activity of catechin is thought to be due to the catechin action on the plasma membrane of the target cell7. The accumulated results analyzing the effect of catechin on the cell membrane fluidity have most obtained information from one region (or average) using single molecular probes for bulk membrane fluidity. However, the membrane fluidity may be different at positions.
The objective of this study was providing a foundation for studying the mechanism on action of catechin. We carried out a comprehensive study of action of catechin on the Porphyromonas gingivalis outer membranes (OPGs). The scope of this research is as follows. First, we investigated the effect of catechin on the range and rate of lateral mobility of bulk OPGs lipid bilayer using intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py). Second, we evaluated the effect of catechins on the bilayer lateral mobility of OPGs lipds using fluorescence quenching method specifically developed for the study to measure the range and rate of asymmetrical lateral mobility between inner and outer monolayers of the lipid bilayer, we assessed the effect of catechin on the transbilayer lateral mobility of the OPGs lipid bilayer. Third, we examined effects of catechin on both protein distribution and annular lipid fluidity in OPGs. Fourth, by employing 1,6-diphenyl-1,3,5-hexatriene (DPH), we investigated the effect of catechin on the range of rotational mobility of bulk OPGs lipid bilayer and we examined the rate of asymmetrical rotational mobility between the inner and outer monolayers of the OPGs lipid bilayer.
Ⅱ. MATERIALS AND METHODS
1. Matarials
The fluorescent probes, Py-3-Py, and DPH were purchased from Molecular Probes, Inc. (Junction City, OR, USA). Bovine serum albumin (BSA), catechin and N-2-Hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (Hepes) were obtained from Sigma Chemical (St. Louis, MO, USA). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was purchased from Fluka (Switzerland). All other reagents were obtained commercially with the highest quality available. Water was used after deionized.
2. Bacterial growth conditions
ATCC 33277 (PG) was purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured as stated previously13. PG was maintained as frozen stock cultures and grown in anaerobic environment (Bactron Ⅳ Anaerobic Chamber, Sheldon Manufacturing Inc., OR, USA) in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 1% (wt/vol) yeast extract, 0.0005% (wt/vol) hemin, and 0.0001% (wt/vol) menadione at 37°C for 3 days in an atmosphere of 80% N2, 10% CO2, and 10 % H2.
3. Isolation of outer membrane
The method was proceed by Smalley et al. (1993)14. Briefly, lyophilized whole cells (200 mg) were suspended in 60 ml of 0.14 M NaCl containing 10 mg of EDTA (pH 7.3) and the suspension was incubated at 37°C for 30 minwith stirring15. This serves to dissociate OPGs and to prevent any possible cell associated protease activity16 during preparation. Subsequent to getting through a 25 gauge needle twice, the cells were harvested by centrifugation (20,000´g for 30 min, 4°C) leaving the supernatant which was crude OPGs preparation. Remaining EDTA and buffer salts were eliminated by dialysis against distilled water for 4 h at 4°C, and OPGs fraction was then freeze-dried. The protein concentration was determined by Lowry et al. (1951)17 using BSA as a standard was followed.
4. TNBS Labelling
To evaluate the fluorescence parameters of probe molecules in each membrane monolayers, the TNBS labeling reactions was performed as described18 with a few modifications. The synaptosomal pellet was softly resuspended in 50 ml of 4 mM TNBS in buffer A for 80 min (for asymmetric lateral mobility) or 40 min in 50 ml of 2 mM TNBS in buffer A (for asymmetric rotational mobility) or in buffer A alone. Buffer A contained 11 mM glucose, 120 mM NaHCO3, 30 mM NaCl and 1% bovine serum albumin (BSA), pH adjusted to 8.5 with NaOH. To ensure that all synaptosomal outer monolayers are completely exposed to TNBS, the pellet was passed at a slow pace through an Eberbach tissue grinder (3 strokes up and down). Unless otherwise specified, treatment was performed at 4°C. The TNBS labeling reaction was finished by adding an equal volume of 1% BSA in phosphate buffered saline (PBS; 0.2 g/l KCl, 8 g/l NaCl, 1.15 g/l Na2HPO4·7H2O, 0.2 g/l KH2PO4, 0.48 g/l Hepes, pH 7.4).
5. Effect of Catechin on the Structure of the Individual Monolayers of OPGs: Selective Quenching of Py-3-Py
The experimental measurement of the structure of the individual monolayer of OPGs is based on the method previously established for the OPGs with a few modification18. The method we used is on the basis of assumption that the system is consists of fluorescent compartments that are differentially accessed by TNBS. The excimer to monomer fluorescence intensity ratios, I'/I, of Py-3-Py in bulk (inner plus outer), and in the inner and outer monolayers were computed from the following equations:
where (I'/I)t, (I'/I)i and (I'/I)o are the excimer to monomer fluorescence intensity ratios of bulk Py-3-Py (I'/I), and in the inner and outer monolayers, respectively. The values of 
(excimer fluorescence intensity for inner plus outer monolayers) and (excimer fluorescence intensity for the inner monolayer) were obtained from OPG incubated with buffer A and buffer A plus TNBS, respectively, for Py-3Py, at 4°C (pH 8.5) (non-penetrating conditions).
6. Determination of Annular Lipid Fluidity in OPGs
The experimental determination of the annular lipid fluidity in OPGs is based on a previously established methods for synaptic plasma membrane19 and OPGs18.
Py-3-Py incorported into SPMVs was excited by radiationless energy transfer (RET) from tryptophan (excitation at 286 nm) and the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py was computed from the ratio 480 nm to 379 nm signal. Considering that the Förster radius (the RET-limiting distance) for the tryptophan-Py-3-Py donor-acceptor pair is 3 nm18, only Py-3-Py located on the annular lipids (close to proteins) was excited, and annular lipid fluidity was considered proportional to I'/I18.
7. Determination of Protein Distribution in the OPGs Llipid Bilayer
This was based on previously established medhods for membranes18. We measured the fluorescence intensity of endogenous tryptophan in OPGs. The Py-3-Py probe was then incorporated at a concentration of 5´10-7 M (in absolute ethanol), and tryptophan emission fluorescence intensity was measured again after 10 min. The RET efficiency from tryptophan to Py-3-Py was computed from the equation:
where Ida and Id stand for the fluorescence intensities of donor (in this case, endogenous tryptophan) in the presence and absence of the acceptor (Py-3-Py), respectively. The excitation and emission wavelengths of tryptophan were 286 and 335 nm.
8. Effect of Catechin on the Rotational Mobility of Bulk OPGs
The intensities of fluorescence components perpendicular (I┴) and parallel (I||) to the direction of the vertically polarized excitation light were determined by quantifying the light emitted through horizontally and vertically oriented polarizers. The polarization (P) was acquired from intensity measurements using P = (I||- GI┴) / (I||+ GI┴) where G is the grating for the transmission efficiency of the monochromator for vertical and horizontal polarizations. It is given as the ratio of the fluorescence intensities of the vertical to horizontal components when the exciting light is polarized in the horizontal direction. The polarization was expressed as the anisotropy [r = 2P/(3-P)] (equation 5).
9. Effect of Catechin on the Separate Monolayers of OPGs: Selective Quenching of DPH
The experimental determination of monolayer structure isolated from OPGs is based on a method developed by Schroeder and Kinden (1980)20 for tumor cell plasma membranes. Rather than simply providing theoretically calculated values or average values, it is based on the assumption that the system consists of fluorescent compartments with different accessibility to TNBS. If anisotropy, r and fluorescence intensity F are measured simultaneously,
where Fj is the fraction of fluorescence intensity in compartment j. For a binary system constituted of the outer and inner monolayers of the SPMVs, this leads to
where F and Fi are the DPH fluorescence obtained for SPMV incubated with buffer A and buffer A plus 2 mM (for 40 min) TNBS at 4ºC (pH 8.5) (non-penetrating conditions), respectively. The values of fluorophore concentration- independent parameter anisotropies, r (anisotropy for both monolayers) and ri (inner monolayer anisotropy), were also settled for DPH in SPMVs incubated with buffer A and buffer A plus TNBS respectively at 4ºC. The equation was then solved for ro (outer monolayer anisotropy).
10. Fluorescence Measurements
The fluorescence mensurations were conducted using a modified method of previous studies18. The OPGs were suspended in PBS at a concentration of 50 μg protein/ml.
All fluorescence was measured with a Multi Frequency Cross-Correlation Phase and Modulation Fluorometer (Model; ISS K2-003). Cuvette temperature was maintained at 37.0 ± 0.1°C in a circulating water bath (pH 7.4). Bandpass slits were 10 nm for excitation and 5 nm for emission. A blank prepared under the same conditions without fluorescent probes was used as control.
Py-3-Py was incorporated by adding aliquots of a 5´10-5 M stock solution in absolute ethanol to the OPGs so that the final probe concentration was less than 5´10-7 M18. Mixtures were first vigorously vortexed at room temperature for 10 s and then incubated at 4°C for 18 h with gentle stirring 18.
DPH was dissolved in tetrahydrofuran, and 0.5 μl tetrahydrofuran per ml of PBS was added directly to the membrane suspension to a concentration of 0.01 μg/50 μg membrane protein (fluorescent probe DPH 2: membrane protein 10,000) as previously described 18. After probe incorporation, the membrane suspension was loaded into a cuvettes and control fluorescence was determined. A concentrated solutions of catechin was prepared in 10 mM Tris-HCl (pH 7.4) and added to the labeled membrane suspension (or untreated OPGs suspension) to obtain the desired concentration of catechin (in this case, 30 min incubation).
Wavelengths of excitation were 330 nm for Py-3-Py and 286 nm for tryptophan. Wavelengths of emission were 480 nm for Py-3-Py excimer, 379 nm for Py-3-Py monomer and 335 nm for tryptophan. A GG-455 cut-off filter was used for Py-3-Py excimer emission. The excimer to monomer fluorescence intensity ratio, I'/I, was calculated as the 480 nm to 379 nm signal ratio. The excitation wavelength of DPH was 362 nm and the emission wavelength was 424 nm.
Ⅲ. RESULTS
1. Effects of Catechin on the Rate and Range of Lateral Mobility in Bulk Bilayer OPGs
The I'/I value in intact OPGs (untreated with catechin) was 0.593 ± 0.010 (at 37°C, pH 7.4, Table 1). Incubation with catechin increased the rate and range of lateral mobility of bulk (inner + outer monolayer) OPGs at concentrations as low as 100 μM (n = 5, P < 0.05), as shown in Fig. 1.
Catechin significantly increased the range and rate of lateral mobility of the outer monolayer to a significant extent (0.660 ± 0.010, n = 5, P < 0.05) at 50 μM catechin (Fig. 1). It had a greater efficacy on the lateral mobility of the outer monolayer (Fig. 1, filled triangles) than nner monolayer (Fig. 1, filled circles). Since the change in I´/I values are mainly due to effects on the outer monolayer, we studied the selective effect of catechin on the rate and range of lateral mobility. The I´/I value of intact OPGs outer monolayer (catechin untreated) was 0.649 ± 0.018 (at 37°C, pH 7.4). Above 50 μM (n = 5, P < 0.05) concentration, the range and rate of lateral mobility of OPGs outer monolayer significantly increased (Fig. 1). The I´/I value of Py-3-Py in OPGs incubated with 300 μM was 0.758 ± 0.015 (n = 5, P < 0.01, at 37°C, pH 7.4), compared to before addition of 300 μM. The change in I´/I value before additon was 0. 109. The Py-3-Py values of the outer monolayer were 0.649 ± 0.018 (n = 5) and 0.534 ± 0.015 (n = 5) at 37 and 25°C (pH 7.4), respectively.
Thus the effect of 300 μM catechin was equivalent to the effect produced by a temperature increase of approximate 11.4°C. To the best of our knowledge, the results for asymmetric lateral mobility shown on this paper are the first to prove that the Sheetz-Singer hypothesis (1974)21 is valid in bacterial outer membranes.
2. Effects of Catechin on the Range and Rate of Transbilayer Asymmetric Lateral Mobility of OPGs Monolayers
The effect of increasing concentrations of catechin on the I'/I values in the individual OPGs monolayers is shown in Fig. 1. Catechin increased the rate and range of lateral mobility of the outer monolayer to a significant extent (0.660 ± 0.010, P < 0.05, n = 5) at 50 μM catechin (Fig. 1). Catechin increased the fluidity of the outer monolayer (Fig. 1, filled triangles) more significantly than inner monolayer (Fig. 1, filled circles). Since the changes in I'/I values were mainly derived from effects on the outer monolayer, we studied the selective effects of catechin on the rate and range of probe's mobility. To the best of our knowledge, the results shown on this paper are the first to prove that the Sheetz-Singer hypothesis (1974)21 is valid in bacterial outer membranes.
3. Effects of Catechin on Annular Lipid Fluidity in the OPGs Lipid Bilayer
I´/I measurements showed that the annular lipid fluidity in OPGs (intact outer membrane) was 0.320 ± 0.013 (n = 5, at 37°C, pH 7.4), which increased in response to concentration of 50 μM catechin and obove (Fig. 2).
The I´/I values of Py-3-Py in the bilayer are 0.320 ± 0.013 (n = 5) and 0.269 ± 0.012 (n = 5) at 37 and 25°C, respectively. Thus the effect by 300 μM catechin was the equivalent to that produced by a temperature increase of 12.9°C.
4. Effects of Catechin on Protein Distribution in OPGs−
Protein distribution by RET from tryptophan to Py-3-Py was evaluated. The RET value of untreated OPGs was 0.223 ± 0.003 (37°C, pH 7.4), which was lowered by cathchin concentrations above 50 μM (Fig. 3). Protein clustering is probably caused by interactions between phospholipids, especially annular lipids, whose movement is increased by catechin and proteins around them.
5. Effects of Catechin on the Range of Rotational Mobility of Bulk Bilayer OPGs
The anisotropy (r) of DPH in the intact OPGs was 0.138 ± 0.002 (at 37°C, pH 7.4, Table 1). Catechin increased rotational mobility with a significant decrease in anisotropy (r) even at 100 μM or above (Fig. 4). The difference in anisotropy of the bulk OPGs lipid bilayer before and after adding 300 μM catechin was 0.021. This can be evaluated by comparing the effect of temperature on this parameter. The anisotropy of DPH in the bilayer was 0.138 ± 0.002 (n = 5) at 37°C and 0.174 ± 0.002 (n = 5) at 25°C (pH 7.4). Thus, the effect of 300 μM catechins was equivalent to that produced by a temperature increase of approximate 7.0°C.
6. Effects of Catechin on the Range of Transbilayer Asymmetric Rotational Mobility of OPGs Monolayers
The structures of intact OPGs (inner plus outer monolayers), outer (extracellular) and inner (intracellular) monolayers were evaluated using DPH as fluorescent reporter and trinitrophenyl groups as quencher. Trinitrophenylation of the intact OPGs at 4°C (non-penetrating conditions) results in covalent attachment of trinitrophenyl quenching agents to the outer monolayers. About half of the DPH fluorescence was quenched in the outer monolayer of treated OPGs. When TNBS labeling was performed under infiltration conditions (37°C), the fluorescence of the DPH was quenched by more than 90%. Fluorescence parameter values of intact OPGs (both monolayers) and TNBS-treated OPGs (inner monolayer) are listed in Table 1. The DPH anisotropy of the inner monolayer was 0.019, which was much greater than that calculated for the outer monolayer (Table 1).
Fig. 4 shows that the anisotropy of DPH in TNBS-untreated membrane (inner plus outer monolayers) decreased gradually (fluidization) with increasing catechin concentrations (Fig. 4, filled squares). There was a similar but more gradual lessening in the computed anisotropy of the outer monolayer (Fig. 4, filled triangles). However, there was no statistically significant decline in the anisotropy (range of rotational mobility) of the inner monolayer at the catechin concentration of 50 μM used. These results indicate that the fluidizing effect (range of rotational mobility) of catechin is selective.
The anisotropy of DPH in the outer monolayer was 0.129 ± 0.002 (n = 5) at 37°C and 0.162 ± 0.002 (n = 5) at 25°C (pH 7.4). The difference in anisotropy of the outer monolayer before and after adding 300 μM catechin was 0.028. Thus, the effect of 300 μM catechin was equivalent to that produced by a temperature increase of approximately 10.2°C.
The catechin used in this study did not show a significant increasing effect of lateral and rotational mobility on the inner monolayer as well as the bulk bilayer of the bacterial outer membrane at concentration of 100 μM, it was confirmed that it did not show a significant increasing effect. Mobility of the the two types of outer monolayer at a concentration of 50 μM. However, it was confirmed that the mobility in the outer monolayer incresed significantly at a concentration of 50 μM, whereas the mobility in bulk OPGs increased significantly at a concentration level of 300 μM, which is 6 times higher than the concentration for the outer monolyer. Thus, catechin has a selective fluidity effect within the trasnbilayer domains of the OPGs.
Ⅳ. DISCUSSION
We used Py-3-Py, a pyrene derivative used to quantify lateral mobility within OPGs18, to determine the rate and range of lateral mobility in the OPGs. With this probe, the emission of both the monomer (I) and the excimer (I´) components is monitored in such a way that a ratio can be derived and used as a measure of lateral mobility18. Emission from the excimer predominates as probe mobility increses. Because the formation of an intramolecular excimer is dependent upon lateral movement of its two components. Thus, an increase in the I´/I ratio implies increased lateral mobility of the probe within the membranes. The excimer fluorescence technique employing Py-3-Py has the benefit over its counterpart on the basis of intermolecular excimerization in that immensely low probe concentrations (<10-7 M) can be used and perturbation of the OPGs by the probe molecule is minimized.
The covalently lbound trinitrophenyl group has a broad absorbance range colse to 420 nm maximum. This peak largely overlap with the fluorescence emission of Py-3-Py. This overlap is partly responsible for the high transfer (quenching) effectiveness of the probe. Approximately half of the Py-3-Py fluorescence was quenched in trinitrophenylated OPGs. Almost 90% of the Py-3-Py fluorescence was quenched when TNBS labeling was performed under penetrating conditions (37°C). Values of the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py in intact OPGs (both monolayers) and in TNBS-treated OPGs (inner monolayer) are in Table. 1. The I´/I of Py-3-Py in the outer monolayer was 0.113, which was greater than the value calculated for the inner monolayer. This indicates that the range and rate of lateral mobility of the outer monolayer is larger than that of the inner monolayer.
Despite the fact that many researchers have reported that the inner and outer monolayers of and model native membranes have different fluidity, all former studies of asymmetric bilayer fluidity have investigated the rotational range but not the range and rate of lateral mobility. In this study, we investigated transbilayer asymmetric fluidity employing the selective quenching of Py-3-Py and DPH fluorescence by trinitrophenyl groups.
It is significant to note that the term "membrane fluidity" is used in improper way frequently. It arose from a combination of spectroscopic studies, the realization that membranes can be considered as two-dimensional fluids, and the desire to obtain a simple single physical parameter that describes the properties of membranes. The difficulty with the concept of membrane fluidity is that any selected physical parameter will be a function of the spectroscopic method used, in particular the specific time window and the properties of the probe (charge, shape, location etc) 22. The concept of membrane fluidity also is contingent upon the assumption that the hydrophobic region of cell membranes is dynamically and structurally homogeneous, and this assumption is currently being severely challenged. So, while it might be true to state that the bulk or average spectroscopic properties of cell membranes is probably not useful for hypothesizing the pharmacological action(s) of drug(s), local properties related to domains or immediate environment of a membrane protein may be very relevant.
As already mentioned, membrane bilayer mobility is one of the significant factors controlling membrane fluidity or microviscosity. It is well known the membrane bilayer mobility includes lateral mobility, rotational mobility and flip-flop, the most important of which is lateral mobility. We are pleased to be the first to develop and report a fluorescence quenching technique capable of measuring membrane transbilayer lateral mobility. Therefore, we assume that this study will conduce to drug-membrane interaction studies.
The explicit mechanism of action of the drug on the enhanced effects of SPMV on annular lipid fluidity is unknown. However, the mechanism by which catechin increases the annular lipid fluidity of the SPMV lipid bilayer can be hypothesized as follows.
Annular lipids are well known to enclose proteins with or without physical association with them. Catechin can bind to lipids, especially annulr lipids, increase the mobility of proteins and indirectly affect the dynamic behavior of proteins, thereby altering the conformational structure or dynamics of these proteins. Because biological membranes are highly complex in composition, it has been unfeasible to monitor alterations in the local lipid environment and simultaneously determine their effect of catechin on membrane protein function. Nevertheless, it is likely that the spotted effects are not only caused by the effects of catechin on lipids, but also enhanced by the interactions between lipids and proteins.
Thus, catechin affect the lateral and rotational mobility of OPGs primarily through their effect on the outer monolayer of the OPGs. This is the first demonstration that catechin differentially affect transbilayer rotational and lateral mobility of the inner and outer monolayers of OPGs. OPGs, specifically the outer monolayer, appears to be much more sensitive to the fluidization effect of catechin, and this discovery can be extended to the bilayer asymmetric fluidity of OPGs.
From the results of our study, there is no doubt that catechin increased lateral and rotational mobility of the OPGs lipid bilayer. Catechin which increase lateral and rotational mobility of the OPGs lipid bilayer mostly increased the mobility of outer monolayer. These effects are not only due to the effect of catechin on lipids, but also enhanced by interaction between lipids and proteins. This conclusion can be deduced because we verified that the magnitude of the effect of the increase in annular lipid fluidity in SPMV lipid bilayer induced by catechin was much greater than the magnitude of the increase in effect of the drug on the lateral and rotational mobility of bulk OPGs lipid bilayer.
Opinions have been divided as to whether catechin directly bind to proteins and interfere with membrane protein functions, of whether the main mechanism of action occurs indirectly through changes in the physicochemical characteristics of the lipid membranes through which catechin diffuse easily. Because biological membranes are of highly complex in composition, it has been unfeasible to monitor alterations in the local lipid environment and simultaneously determine effect of catechin on the membrane protein function. The proliferative effect canbe explined by citing lipid-induced increases in the mobility of proteins, but the opposite case, in which proteins induce changes in lipids, is more likely. Although it is clear that catechin increase bacterial outer membrane mobility, the direct effect of catechin on protein appears to magnify such effects onf lipids. That is, fluidization of membrane lipids before, during, or after the interaction of the catechin with proteins can provide an ideal microenvironment for optimum antimicrobial effects. In conclusion, our data suggest that, in addition to direct interaction with membrane phospholipids, catechins affect the fluidity of the bacterial outer membranes by simultaneously interacting with membrane proteins.
