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Interactions of Genistein and Related Isoflavones with Lipid Micelles William L. Whaley,* Jeremy D. Rummel, and Niksa Kastrapeli Chemistry Department, Texas A&M UniVersity-Commerce, Commerce, Texas 75429 ReceiVed March 9, 2006. In Final Form: May 24, 2006 Genistein (5,7,4′-trihydroxyisoflavone) modulates the function of several transmembrane ion-channel proteins by mechanisms that are unrelated to phosphorylation events. Daidzein (7,4′-dihydroxy-isoflavone) typically exhibits modest effects, whereas genistin (7-O-glucosyl-genistein) usually exhibits no effect on ion-channel activities. Genistein appears to modulate gramicidin A ion channels by alteration of bilayer mechanical properties, but the associated molecular interactions have not been defined. The incorporation of daidzein into phosphatidylcholine liposomes promotes aggregation and precipitate formation which is problematic for structural studies based on NMR spectroscopy. In the present study, daidzein was incorporated into sodium dodecyl sulfate (SDS) micelles to provide a stable system with no evidence of micelle aggregation. For this reason genistein, daidzein, genistin, and osajin (a diprenyl-genistein derivative) were incorporated into SDS micelles (in D2O) to evaluate differences in position and orientation within micelle structures. The 1H NMR line widths, as a function of Mn2+ concentration, indicate that genistein is quite mobile and buried within the hydrophobic micelle core. Daidzein and genistin also are mobile but exhibit average positions near the micelle/aqueous interface, with polar groups oriented toward the aqueous compartment. These results demonstrate that daidzein, with only two hydroxyl substituents, has a greater affinity for a polar environment than genistein with three hydroxyl substituents. The 5-hydroxyl group of genistein forms an intramolecular hydrogen bond with the 4-carbonyl group, which diminishes the molecular affinity for a polar matrix. These results suggest an explanation for the relative abilities of these compounds to increase gramicidin channel lifetimes and modulate other ion-channel types.
Introduction Genistein is one of the most studied isoflavones,1,2 and there is evidence that many of its biochemical activities are due to modulation of protein kinase and lipid kinase signaling cascades.3 Genistein is a tyrosine kinase inhibitor4 and modulates the activities of some transmembrane ion channels by protein phosphorylation or by direct interaction with specific amino acid sequences.5 Other functional effects of genistein are less specific. The lifetimes of gramicidin A ion channels, in planar lipid bilayers, were increased 7-fold by the presence of 40 µM genistein.6 Daidzein exhibited a modest effect, whereas genistin had no effect on gramicidin A ion-channel lifetimes. The effects of these isoflavones, on conductance parameters of synthetic gramicidin ion channels of different lengths in bilayers of different thicknesses, demonstrated that genistein increased ion-channel lifetimes by alteration of bilayer mechanical properties and not by specific interactions with the peptide structure. Genistein was demonstrated to inhibit voltage-dependent sodium and potassium channels at micromolar concentrations, whereas daidzein exhibited moderate inhibition and genistin had a very weak effect.7 Genistein has exhibited strong modulation of many types of ion channels with very different amino acid * E-mail:
[email protected]. (1) Dixon, R. A.; Ferreira, D. Genistein. Phytochemistry 2002, 60 (3), 205211. (2) Birt, D. F.; Hendrich, S.; Wang, W. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol. Ther. 2001, 90, 157-177. (3) Williams, R. J.; Spencer, J. P. E.; Rice-Evans, C. Flavonoids: Antioxidants or Signaling Molecules? Free Rad. Biol. Med. 2004, 36 (7), 838-849. (4) Akiyama, T.; Ishida, J.; Nakagawa, S.; Ogawara, H.; Watanabe, S.; Itoh, N.; Shibuya, M.; Fukami, Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 1987, 262 (12), 5592-5595. (5) Illek, B.; Fischer, H.; Santos, G. F.; Widdicombe, J.; Machen, T. E.; Reenstra, W. W. cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein. Am. J. Physiol. 1995, 268 (4 pt 1), C886-893. (6) Hwang, T. C.; Koeppe, R.; Andersen, O. Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics. Biochemistry 2003, 42 (46), 13646-13658.
sequences, whereas daidzein has typically exhibited modest effects and genistin has exhibited little or no effects.6,7 These results suggest that genistein modulates ion channels by mechanisms not involving interaction with specific peptide sequences. The actions of genistein on ion channels may therefore depend on its interaction with lipid molecules closely associated with the peptide structure. The effects of genistein, daidzein, and genistin on the conductance properties of gramicidin A, voltage-gated sodium, and voltage-gated potassium channels were correlated with assumed relative hydrophobicities of these compounds to support mechanistic arguments.6,7 Daidzein routinely has been assumed to be more hydrophobic than genistein based on the number of hydroxyl substituents. This assumption was incorrect, and mechanistic arguments should be revised using measured parameters as indices of molecular polarity. In the case of gramicidin ion channels, genistein appears to facilitate mechanical stress due to hydrophobic mismatch imposed by the formation of conducting dimers.6 It also seems possible that genistein, by interacting with lipid molecules in close contact with voltage gated sodium and potassium channel proteins, could also inhibit these channels as well.7 These proposed mechanisms would require genistein to adopt an average position and orientation within the bilayer that facilitated interaction with the lipid molecules affected by movement of protein domains. The observation that genistin is usually inactive might be explained by the very polar glucosyl group restricting this molecule to the aqueous interface in an orientation that is improper to alter bilayer mechanical properties associated with channel function. The observation that daidzein is usually moderately active could be due to its intermediate tendency to interact with the membrane aqueous interface. In (7) Liu, L.; Yang, T.; Simon, S. A. The protein tyrosine kinase inhibitor, genistein, decreases excitability of nociceptive neurons. Pain 2004, 112, 131141.
10.1021/la0606502 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/18/2006
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Figure 1. Chemical structures of studied compounds with proton assignments. The structures of genistein and related isoflavones are illustrated with the A, B, and C rings designated for each compound. The hydrogens are labeled according to the carbons they are attached to using standard nomenclature. The protons of the isoflavone nucleus for osajin are labeled by standard nomenclature and the protons of the isoprenyl groups are labeled as a continuation of this numbering system for simplicity. These proton designations are used for all discussions regarding 1H NMR spectroscopy in the text.
addition, the relative tendencies of isoflavones to partition into lipid structures must also be considered when interpreting experimental data to formulate mechanistic arguments. Since genistein is amphiphilic, it has often been assumed to localize at the aqueous interface of membranes; however, a report based on fluorescence anisotropy has indicated genistein and related isoflavones are localized in the hydrophobic core of vesicles.8 Nuclear magnetic resonance (NMR) spectroscopy is also a valuable technique for examining the positions of peptides and small molecules within membranes and lipid structures.9,10 For protons in solution phase, the 1H NMR signal width is proportional to the spin lattice relaxation rate (1/T1), which in the presence of Mn2+ is dominated by the paramagnetic mechanism, and related to 1/r6 as given by the SolomonBloembergen equation.11 The structures of the isoflavones used in this study are illustrated in Figure 1. Genistein has a hydroxyl group at position 5, that is not present in daidzein, which forms a very strong intramolecular hydrogen bond with the carbonyl group at position 4. The hydrogen bond effectively diminishes the partial negative charge on the carbonyl oxygen atom and partially blocks the carbonyl group from dipolar interactions with other polar molecules. This explains the fact that genistein has longer retention times on reversed-phase HPLC columns than daidzein.2 In the present study, genistein and related isoflavones were compared with respect to 1-octanol/water partition coefficient (Kp), position in aqueous SDS micelles, and chromatographic retention time (tR) to better understand the molecular interactions responsible for the observed differences in ion-channel modulation. (8) Arora, A.; Byrem, T. M.; Nair, M. G.; Strasburg, G. M. Modulation of Liposomal Membrane Fluidity by Flavonoids and Isoflavonoids. Arch. Biochem. Biophys. 2000, 373 (1), 102-109. (9) James, T. L. In Nuclear Magnetic Resonance in Biochemistry; Academic Press: New York, 1975; pp 413. (10) Lindberg, M.; Graslund, A. The position of the cell penetrating peptide penetratin in SDS micelles determined by NMR. FEBS Lett. 2001, 497 (1), 3944.
Small unilamellar vesicles composed of phosphatidylcholine (PC) have been used to study flavonoid compounds; however, daidzein was reported to promote PC vesicle aggregation resulting in precipitate formation, whereas genistein did not have this effect.12 Interestingly, formononetin (4′-O-methyl-daidzein) also promoted liposome aggregation, whereas prenylated isoflavones, including 6,8-diprenylgenistein, exhibited a reduced tendency to promote vesicle aggregation.13 In the present study, it was found that incorporation of 1 mM daidzein into lysophosphatidylcholine (LPC) micelles also caused aggregation and precipitate formation. In contrast, incorporation of 1 mM daidzein into aqueous sodium dodecyl sulfate (SDS) micelles produced solutions with stable absorption spectra that were free of precipitate for several weeks. Genistein, genistin, and osajin were also incorporated into aqueous SDS micelles to produce stable solutions. The 1H NMR spectra for the aromatic regions of isoflavones incorporated into SDS micelles are illustrated in Figure 2. An assay based on measurement of 1H NMR signal line widths at one-half-height (∆ν1/2h) as a function of the aqueous Mn2+ concentration was used to demonstrate that subtle structural variations can dictate significant differences for isoflavone position and orientation in SDS micelles. Materials and Methods i. Commercially Obtained Reagents. Inorganic salts were ACS grade with lead(II) acetate obtained from J. T. Baker and manganese chloride (MnCl2) from Mallinckrodt Chemicals. Diethyl ether, ethanol, glacial acetic acid, and xylene were all ACS grade solvents (Mallinckrodt or J. T. Baker). Methanol, acetonitrile, 1-octanol, and water were HPLC grade solvents (EMD Chemicals, Inc.). Sodium (11) Bloembergen, N. In Nuclear Magnetic Relaxation; W. A. Benjamin: New York, 1961. (12) Lehtonen, J. Y.; Adlercreutz, H.; Kinnunen, P. K. Binding of daidzein to liposomes. Biochim. Biophys. Acta 1996, 1285 (1), 91-100. (13) Hendrich, A. B.; Malon, R.; Pola, A.; Shirataki, Y.; Motohashi, N.; Michalak, K. Differential interaction of Sophora isoflavonoids with lipid bilayers. Eur. J. Pharm. Sci. 2002, 16, 201-208.
Position of IsoflaVones in Lipid Micelles
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Figure 2. Comparison of the 300 MHz 1H NMR spectra of genistein, daidzein, genistin, and osajin incorporated into SDS micelles. Isoflavones were incorporated into 250 mM SDS/D2O (with 300 mM H2O) at the following concentrations: genistein (1.3 mM), daidzein (0.8 mM), genistin (0.8 mM), and osajin (0.8 mM). A sample of 2.0 mM osajin was prepared by placing the 0.8 mM osajin sample in a screw-topped centrifuge tube and adding a small amount of solid osajin and sonicating for a second period. Each FID was acquired with 256 transients and processed with a line broadening factor of 0.2 hz. Significant line broadening was observed for the 2 mM osajin sample with two signals observed for H2 (2a and 2b). The broad signal (2b) is more pronounced in the 2 mM osajin sample and is the dominant feature in the spectrum for 5 mM osajin in 250 mM SDS/D2O (data not illustrated). dodecyl sulfate (SDS), deuterated SDS (d25-SDS), DMSO-d6 (99.9% D), and D2O (99% D & 99.9%D) were obtained from Sigma-Aldrich. Genistein, daidzein, and genistin were obtained from Indofine Chemical Company. ii. Preparation and Analysis of Osajin. Osajin was extracted from the dried and ground fruit of Maclura pomifera (Osage orange) using a protocol based on 1/20 the scale of the originally published procedure.14,15 Briefly, 60 g of powdered tissue was extracted with 500 mL of low-boiling point petroleum ether for 48 h using a Soxhlettype extractor. A second extraction with diethyl-ether was done for 16 h to collect a mixture of osajin and pomiferin in roughly equal amounts. The ether was removed by rotary evaporation, and the residue was dissolved in 300 mL of hot 95% ethanol. Pomiferin was precipitated by the addition of 30 mL of 0.10 M Pb (II) acetate (in methanol) and removed by vacuum filtration. The filtrate (containing osajin) was collected, and the solvent was removed by rotary evaporation. The residual solid osajin was recrystallized twice in 95% ethanol, vacuum desiccated, and verified by melting point (187189 °C)16 and 1H NMR spectroscopy. (14) Walter, E. D.; Wolfrom, M. L.; Hess, W. W. A Yellow Pigment from the Osage Orange (Maclura pomifera Raf.). J. Am. Chem. Soc. 1938, 60, 574-577. (15) Wolfrom, M. L.; Mahan, J. Osage Orange Pigments. IX. Improved Separation: Establishment of the Isopropylidene Group. J. Am. Chem. Soc. 1942, 64, 308-311. (16) Wolfrom, M. L.; Harris, W. D.; Johnson, G. F.; Mahan, J. E.; Moffett, S. M.; Wildi, B. Osage Orange Pigments. XI. Complete Structures of Osajin and Pomiferin. J. Am. Chem. Soc. 1946, 68, 406-418.
iii. Analysis and Purification of Osajin by HPLC. The purity of osajin preparations was assessed by isocratic high performance liquid chromatography (HPLC) using a palmitamidopropylsilane column (RPC16-amide, 1.0 cm × 25.0 cm, Supelco) and a mobile phase of 90% methanol/10% acetonitrile delivered at a flow rate of 3.0 mL/minute. The instrument was assembled from components: (1) pump, Spectraphysics SP8700 XR; (2) manual injector valve, Valco; (3) reverse flushing valve, Rheodyne; (4) absorption detector (set at 275 nm), Shimadzu SPD-10 AV. Injections approaching 1 mg total isoflavone allowed baseline resolution of osajin (tR ) 5.50 min) and pomiferin (tR ) 3.20 min). iv. Absorption Spectrophotometry. The absorbance spectra of genistein and osajin were examined in methanol and 250 mM SDS/ D2O solution at 22 °C using a Shimadzu UV 2401-PC spectrophotometer. Isoflavone stock solutions (0.1 mM) were diluted in methanol to obtain an absorbance of 1.0 at the maximum absorbance (λmax) values of 260 nm (genistein) and 274 nm (osajin). A solution of uniform micelles was prepared by briefly sonicating 250 mM SDS in D2O using a cup horn (Bransonic) equipped with recirculating bath (25 °C). An aliquot of 1.5 mL of 250 mM SDS/D2O and 5 mg of isoflavone was placed in a capped tube. Maximum isoflavone incorporation into 250 mM SDS/D2O was achieved by sonication using 30 s bursts interspersed with 30 s cooling periods for a total time of 30 min. Insoluble solids were removed by centrifugation (14 000g, 20 min, 22 °C). This provided solutions of 4 mM genistein and 5 mM osajin in 250 mM SDS/D2O which were diluted with 250
7178 Langmuir, Vol. 22, No. 17, 2006 mM SDS/D2O to an absorbance of 1.0 at the respective λmax values. A solution of 1.0 mM osajin in 250 mM SDS/D2O was prepared by limiting the total sonication period to 10 min. Solid osajin was removed by centrifugation, and the supernatant was diluted with 250 mM SDS/D2O (A274 ) 1.0). v. Determination of 1-Octanol/Water Partition Coefficients. The 1-octanol/water partition coefficients (Kp) were determined by the “shake-flask” method using a published protocol based on absorbance measurement of the aqueous phase.17 Aqueous 50 mM MOPS (pH ) 7.4) was equilibrated at 22 °C with an equal volume of 1-octanol, and phases were separated just prior to the assay. Each isoflavone was dissolved in the aqueous phase to provide a solution with an absorbance of 1.0 at λmax. Five aliquots of 1-octanol were added sequentially to 10.0 mL of the isoflavone solution, and after each addition, the phases were mixed by shaking and separated by centrifugation (4000g, 22 °C, 5 min). The aqueous phase was transferred to a jacketed cuvette (22 ( 0.5 °C), and the absorbance at λmax was recorded. vi. Correlation of log Kp Values with Chromatographic Retention on RPC16-Amide and IAM Columns. A Dionex Summit HPLC system was used with two different column adsorbent/mobile phase systems. An RPC16-amide column (25.0 cm × 0.4 cm, Supelco) was subjected to isocratic elution with 90% acetonitrile/ 10% methanol delivered at 1.000 mL/min. An immobilized artificial membrane (IAM) column (3.0 cm × 4.6 mm, IAM.PC.DD.2, Regis Technologies, Inc.) was subjected to isocratic elution with 100% acetonitrile delivered at 1.000 mL/min. Aliquots of 20 µg of each isoflavone were manually injected onto the HPLC system. Daidzein, genistein, genistin, and osajin were detected at wavelengths of 250, 260, 260, and 274 nm, respectively. Initially, aqueous 0.10 M phosphate buffer (pH ) 7.4) was used as the mobile phase for the IAM column; however, none of the isoflavones would elute. A mixture of 60% acetonitrile/40% aqueous 0.10 M phosphate buffer (pH ) 7.4) also failed to elute these compounds. The IAM column was washed with pure H2O, and a gradient was run to 100% acetonitrile, after which, each of the isoflavones eluted within 20 min. vii. NMR Spectroscopy of Isoflavones in DMSO-d6. Samples of 20 mg of desiccated isoflavone dissolved in 0.7 mL of DMSO-d6 (99.9% D, Cambridge Isotopes) with 1% TMS were analyzed in 5 mm tubes. Spectra were obtained at 22 °C with deuterium signal lock using a Varian 300 MHz Mercury VX NMR Spectrometer with VNMR6.1c software. Signal assignments were made using published data for genistein in DMSO-d618,19 and osajin in acetone-d618 along with 2D-COSY and selective difference NOE experiments (performed on a Varian 400 MHz INOVA NMR with field gradients). viii. Incorporation of Isoflavones into SDS Micelles and Analysis by 1H NMR Spectroscopy. A solution of 250 mM SDS in D2O (containing 300 mM H2O) was sonicated using the cup horn system at 25 °C with 2 min bursts interspersed with 1 min cooling intervals for 20 min. About 5 mg of solid isoflavone was added to 1.5 mL of 250 mM SDS/D2O (with 300 mM H2O) solution. This solution was sonicated using 30 s bursts interspersed with 30 s cooling periods. To obtain maximum incorporation levels, this sonication was carried out for a total time of 30 min. To limit the incorporation of isoflavones to a concentration of about 1 mM, the sonication was performed for a total time of only 10 min. Residual solids were removed by centrifugation (14 000g, 20 min, 22 °C), and supernatants were transferred to thin-wall 5 mm NMR sample tubes and stored at 22 °C. Titration assays were originally performed using samples with maximum isoflavone concentration. After osajin was demonstrated to form multimeric complexes, the assays were repeated with each of the isoflavones at approximately 1 mM concentration. Aliquots (17) Brown, J. E.; Khodr, H.; Hider, R. C.; Rice-Evans, C. A. Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties. Biochem. J. 1998, 330, 1173-1178. (18) Delle Monache, G.; Scurria, R.; Vitali, A.; Botta, B.; Monacelli, B.; Pasqua, G.; Palocci, C.; Cernia, E. Two Isoflavones and a Flavone from the Fruits of Maclura pomifera. Phytochemistry 1994, 37 (3), 893-898. (19) Mabry, T. J.; Markham, K. R.; Thomas, M. B. The Systematic Identification of FlaVonoids; Springer-Verlag: New York, 1970.
Whaley et al. of 1-5 µL of 0.160 M MnCl2 in D2O were added directly to the NMR sample tubes until a final concentration of about 4 mM was reached. The ∆ν1/2h values for SDS proton signals were also measured for solutions of 250 mM SDS/D2O (with 300 mM H2O) titrated with Mn2+. These control experiments were done to verify that incorporation of the isoflavones did not increase the permeability of SDS micelles to Mn2+. 1H NMR spectra (300 MHz) were obtained with deuterium channel lock on D2O and a probe temperature of 22 °C. Acquisitions used a pulse angle of 45° with 256 to 20 480 (20K) transients. Free induction decays were processed using a line broadening factor of 0.2 to 1.0 Hz that was subtracted from measured ∆ν1/2h values. ix. Incorporation of Genistin into Deuterated SDS Micelles Using Mild Conditions. Genistin was incorporated into preformed deuterated SDS (d25-SDS) micelles by simple adsorption. Micelles were prepared by sonication of d25-SDS in D2O as previously described with care given to minimize contamination by H2O vapor. Liquids were transferred by syringe with a needle when possible. Approximately 5 mg of solid genistin was added to 1.5 mL of 250 mM d25-SDS micelle solution in a glass vial with a septum cap. The solution was stirred at 42 °C for 24 h using a magnetic stirrer. The remaining solid particles of genistin were removed by centrifugation (14 000g, 20 min, 22 °C). The clear supernatant, containing 1.5 mM genistin, was transferred to a 5 mm sample tube using a syringe.
Results i. Absorption Spectrophotometry and NMR Spectroscopy of Genistein and Osajin in SDS Micelles. Absorption spectra for samples with different concentrations of osajin in 250 mM SDS/D2O solution were examined to test for self-association of osajin molecules. Genistein could be incorporated at relatively high concentration in 250 mM SDS/D2O and was studied for comparison. A solution of 4 mM genistein in 250 mM SDS/D2O was diluted with 250 mM SDS/D2O solution to an absorbance of 1.0 (at λmax) and immediately scanned. A single symmetric absorption peak was observed (λmax ) 262 nm; 262 ) 56 000 M-1 cm-1). The absorption spectrum for genistein in methanol also exhibited a single symmetric peak (λmax ) 261 nm; 261 ) 56 030 M-1 cm-1). The spectra for genistein in methanol and SDS micelles were nearly identical (see the Supporting Information). A solution of 1.0 mM osajin in 250 mM SDS/D2O was diluted with 250 mM SDS/D2O solution to an absorbance of 1.0 (at λmax). The absorption spectrum exhibited a single peak (λmax ) 278 nm, 278 ) 74 330 M-1 cm-1) similar to that observed for osajin in methanol (λmax ) 274 nm, 274 ) 75 000). A solution of 5.0 mM osajin in 250 mM SDS/D2O was diluted in a similar manner; however, the absorption spectrum was a broad convolution of overlapping components between 260 and 290 nm (see Figure 3). Since osajin has low water solubility (
