Synthesis and n.m.r.-spectral analysis of unenriched and [1-13C]-enriched 5-deoxypentoses and 5-O-methylpentoses

Chemical methods are described for preparing unenriched and [1-13C]-enriched 5-deoxy- and 5-O-methyl-pentoses in the D or L configuration. The 1H-n.m.r. spectra of these compounds have been interpreted, and the 13C-n.m.r. spectra assigned with the aid of 2-D 13C-1H chemical-shift correlation spectroscopy. Tautomeric forms (furanoses, hydrate, and aldehyde) in solution in 2H2O have been quantified with the aid of [1-13C]-enriched derivatives. Spectra of 5-deoxypentoses, 5-O-methylpentoses, and methyl pentofuranosides have been compared, in order to assess the effect of 5-C-deoxygenation and 5-O-methylation on chemical shifts and coupling constants (1H-1H, 13C-1H, and 13C-13C) and on the pentofuranose conformations.     

Interaction of acetazolamide and 4-nitrothiophenolate ion with bivalent metal ion derivatives of bovine carbonic anhydrase.

The stability and rate constants for the interaction of acetazolamide (diamox) and 4-nitrothiophenolate ion (NTP) with the bivalent Mn, Co, Ni, Cu and Cd forms of bovine carbonic anhydrase have been measured by utilizing the distinct visible spectra of each metalloenzyme-NTP adduct. Differing stabilities of the various NTP and (particularly) diamox complexes reside mainly in varying values for the dissociation rate constants (kd). Intrinsic formation rate constants (for the acid form of the enzyme reacting with the basic form of the ligand) are uniformly high (greater than or equal 2 X 10(7) M-1 s-1 at 25 degrees C). Invariance of kd with pH and a bell-shaped log K-pH profile with the Cu-enzyme adducts are features observed previously with the native enzyme. Binding of NTP with the Cu and Cd metalloenzymes is stronger than to the native form.     

Fumarase: viscosity dependence of the kinetic parameters

Fumarase catalyzes the reversible, stereospecific hydration of fumarate to form L-malate. We have determined the viscosity dependence of V/K and V in both the forward and the reverse directions at pH 6.9 in the absence and presence of several viscosogenic reagents. V/K for fumarate hydration decreases with increasing concentrations of glycerol and sucrose, but is unaffected by increasing concentrations of the polymeric viscosogen polyethyleneglycol (av MW, 10,000 da). V/K for malate dehydration similarly decreases with increasing concentrations of both glycerol and sucrose, but is unaffected by increasing concentrations of polyethylene glycol. Equilibrium constants, calculated from the ratio of V/K values for malate dehydration and fumarate hydration at various concentrations of glycerol, closely match the experimentally determined equilibrium constants at the same concentrations of glycerol. Both experimental and calculated equilibrium constants decrease with increasing concentrations of viscosogens. V/K for the dehydration of (-)-tartrate, a poor substrate, is unaffected by increasing concentrations of glycerol. Analysis of the microviscosity dependence of malate dehydration and fumarate hydration suggests that both substrates bind at diffusion-limited rates. The viscosity dependence of substrate and product dissociation steps may also contribute to the viscosity dependence of V/K values for both substrates. The viscosity dependence of the maximal velocities argues that product dissociation steps are rate-limiting and diffusion controlled.     

Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.     

Equilibrium constants for the formation of glyoxylate thiohemiacetals and kinetic constants for their oxidation by O2 catalyzed by l-hydroxy acid oxidase

Glyoxylate thiohemiacetal formation constants (defined as the concentration of thiohemiacetal divided by the concentration of thiol and the total concentration of hydrated and unhydrated glyoxylate) were determined at 25°C and pH 7.4 for a variety of thiols using two independent methods, and were found to be in the range of 0.2 to 1.7 mm^?1. Under the same conditions the hydration constant for glyoxylate (defined as the concentration of the hydrate divided by the concentration of the free aldehyde) was determined to be 163 ± 7. This information is used in conjunction with kinetic data to calculate kinetic constants for the oxidation of the thiohemiacetals by O₂ catalyzed by rat kidney l-hydroxy acid oxidase. The results further indicate that several such thiohemiacetals are excellent substrates, and suggest that one or more of them may be the physiological reactant for this enzyme.     

A novel method for determining rate constants for dehydration of aldehyde hydrates

A method has been developed for calculating rate constants for dehydration of aldehydes that induce ATPase reactions by kinases and where 18O is transferred from the aldehyde or its hydrate to inorganic phosphate during the reaction. The method involves measurement of the fraction of 18O in phosphate by 31P NMR after the ATPase reaction has proceeded for several minutes with zero-order kinetics. The reaction is started by addition of the aldehyde in a small volume of H2 18O, and the speed of washout of 18O by reversible dehydration relative to the rate of the ATPase reaction allows calculation of the rate constants if the hydration equilibrium constant is known from the proton NMR spectrum of the aldehyde. Dehydration rate constants (s-1 at pH 8-8.5, 0.1 M buffer, 25 degrees C) for the following aldehydes (all over 95% hydrated) and kinases used are as follows: D-glyceraldehyde with glycerokinase, 0.03; 2,5-anhydro-D-mannose 6-phosphate with fructose-6-phosphate kinase, 0.025; 2,5-anhydro-D-mannose or 2,5-anhydro-D-talose with fructokinase, 0.029 and 0.017, respectively; D-gluco-hexodialdose with hexokinase, 0.068. With betaine aldehyde and choline kinase or glyoxylate and pyruvate kinase, no 18O was transferred to phosphate during the ATPase reactions. However, the dehydration rate constant for glyoxylate (0.007 s-1 at pH 7 extrapolated to zero buffer concentration and up to 0.11 s-1 at pH 9.0 with 0.3 M buffer) was determined by extrapolating the initial rate of reduction of the free aldehyde catalyzed by lactate dehydrogenase to infinite enzyme levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and alpha- lactalbumin

Direct difference ir spectra are presented as a function of hydration for lysozyme and α-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behaviour of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced “loosening up” of the lysozyme conformation at about 55 mol water/mol protein. α-Lactalbumin appears to “open up” more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.     

The mechanism of addition of pyridoxal 5'-phosphate to Escherichia coli apo-serine hydroxymethyltransferase

Previous studies suggest that the addition of pyridoxal 5'-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5'-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.     

Active site residues and mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-2H]-d-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of kcat is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-2H]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.     

Novel mechanism-based substrates of dihydrofolate reductase and the thermodynamics of ligand binding: A comparison of theory and experiment for 8-methylpterin and 6,8-dimethylpterin

Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of the designed mechanism-based pterins, 8-methylpterin and 6,8-dimethylpterin, to dihydrofolate reductase (DHFR), with co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The calculated free energy differences suggest that DHFR.NADPH.6,8-dimethylpterin is thermodynamically more stable than DHFR.NADPH.8-methylpterin by 2.4 kcal/mol when the substrates are protonated and by 1.3 kcal/mol when neutral. The greater binding strength of 6,8-dimethylpterin may be attributed largely to hydration effects. In terms of an appropriate model for the pH-dependent kinetic mechanism, these differences can be interpreted consistently with experimental data obtained from previous kinetic studies, i.e., 6,8-dimethylpterin is a more efficient substrate of vertebrate DHFRs than 8-methylpterin. The kinetic data suggest a value of 6.6 ± 0.2 for the pK_a of the active site Glu-30 in DHFR.NADPH. We have also used experimental data to estimate absolute values for thermodynamic dissociation constants of the active (i.e., protonated) forms of the substrates: these are of the same order as for the binding of folate (0.1–10 μM). The relative binding free energy calculated from the empirically derived dissociation constants for the protonated forms of 8-methylpterin and 6,8-dimethylpterin is 1.4 kcal/mol, a value which compares reasonably well with the theoretical value of 2.4 kcal/mol. © 1993 Wiley-Liss, Inc.     

Inhibition of cathepsin B by peptidyl aldehydes and ketones: slow-binding behavior of a trifluoromethyl ketone

Inhibition of the cysteine proteinase cathepsin B by a series of N-benzyloxycarbonyl-L-phenylalanyl-L-alanine ketones and the analogous aldehyde has been investigated. Surprisingly, whereas the aldehyde was found to be almost as potent a competitive reversible inhibitor as the natural peptidyl aldehyde, leupeptin, the corresponding trifluoromethyl ketone showed comparatively weak (and slow-binding) reversible inhibition. Evaluation of competitive hydration and hemithioketal formation in a model system led to a structure-activity correlation spanning several orders of magnitude in both cathepsin B inhibition constants (Ki) and model system equilibrium data (KRSH,apparent).     

Sedimentation equilibrium of proteins in density gradients.

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.     

Interaction of tetrahydropteroylpolyglutamates with two enzymes from mitochondria

The dissociation constants of tetrahydropteroylpolyglutamates, having from one to six glutamate residues, have been determined for the two mitochondrial enzymes serine hydroxymethyltransferase and dimethylglycine dehydrogenase. The ratios of the dissociation constants for the mono- and hexaglutamate forms of the coenzyme were 200 and less than 10 for serine hydroxymethyltransferase and dimethylglycine dehydrogenase, respectively. Km and kcat values were determined for the reversible interconversion of serine and glycine as a function of the number of glutamyl residues on the coenzyme. The values in the serine to glycine direction did not significantly change with the number of glutamyl residues, but in the glycine to serine direction, there was a 9-fold increase in the kcat/Km when the longer chain polyglutamates were used as the coenzyme substrate. A sensitive and rapid method for determining the dissociation constants of proteins which bind either tetrahydropteroylpolyglutamates or their 5-methyl and 5-formyl conjugates is described.     

Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase

In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD⁺....Thr-48....R.CH₂OH(H₂>O)....Zn$++,\; stretching\; from\; His-51\; on\; the\; surface\; of\; enzyme\; to\; the\; active\; site\; zinc\; atom\; in\; the\; substrate-binding\; site\; of\; enzyme.\; Further,\; it\; is\; based\; on\; extensive\; studies\; of\; steady-state\; kinetic\; properties\; of\; enzyme\; which\; were\; published\; recently.\; In\; this\; study,\; we\; have\; reported\; the\; pH-dependence\; of\; dissociation\; constants\; for\; several\; competitive\; dead-end\; inhibitors\; of\; yeast\; enzyme\; from\; their\; binary\; complexes\; with\; enzyme,\; or\; their\; ternary\; complexes\; with\; enzyme\; and\; NAD+or\; NADH;\; inhibitors\; include:\; pyrazole,\; acetamide,\; sodium\; azide,\; 2-fluoroethanol,\; and\; 2,2,2-trifluorethanol.\; The\; unified\; mechanism\; describes\; the\; structures\; of\; four\; dissociation\; forms\; of\; apoenzyme,\; two\; forms\; of\; the\; binary\; complex\; E.NAD+,\; three\; forms\; of\; the\; ternary\; complex\; E.NAD+.alcohol,\; two\; forms\; of\; the\; ternary\; complex\; E.NADH.aldehyde\; and\; three\; binary\; complexes\; E.NADH.\; Appropriate\; pK$_a values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.     

Different mechanisms of free fatty acid flip-flop and dissociation revealed by temperature and molecular species dependence of transport across lipid vesicles

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (V(f)) model revealed V(f) values for flip-flop that ranged between approximately 12 and 15 Angstroms(3), with larger values for SUV than for LUV or GUV. V(f) values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.     

A model for circular dichroism monitored dimerization and calcium binding in an EF-hand synthetic peptide.

EF-hand peptides have been shown to bind calcium and dimerize to form an intact protein domain [Shaw, G.S., Hodges, R.S. & Sykes, B.D. (1990). Science, 249, 280-283]. A synthetic 33-residue EF-hand peptide with the sequence of carp parvalbumin CD site demonstrated a seven-fold increase in the apparent calcium dissociation constant with a eight-fold decrease in peptide concentration when fit to a single-site calcium-binding model. This observation is consistent with EF-hand dimerization. This paper describes a method to determine the dimerization dissociation constant and the calcium dissociation constants for both the monomer and dimer forms of this EF-hand peptide using circular dichroism techniques. By monitoring the increase in negative molar ellipticity at 222 nm with increasing peptide concentration under calcium-saturating conditions the dimerization dissociation constant for the synthetic parvalbumin CD site was determined to be 55.68+/-10.76 microM. Using the dimerization constant, the calcium dissociation constants for both the monomer and dimer forms of this peptide were determined by monitoring the change in ellipticity of peptide solutions on addition of increasing amounts of calcium. A fit of this data to a mathematical model that takes into account dimerization results in calcium dissociation constants of 421.3+/-21.56 and 47.06+/-6.72 microM for the monomer and dimer forms, respectively.     

Catalytic activity of Ntau-carboxymethylhistidine-12 ribonuclease: pH dependence.

The pH-dependence of RNAase A and of Ntau-carboxymethylhistidine-12-RNAase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase) catalysis was studied. Apparent acid dissociation constants were obtained by least squares analysis of the kinetics data. These dissociation constants were compared with pKa values of model imidazole compounds, and with pKa values of histidine residues 12 and 119 on the protein. The shapes of the kcat versus pH profiles for RNAase A and its carboxymethyl derivative are very similar, from which it is concluded that the mechanism of catalysis is closely similar in the two proteins. Apparent pKa values obtained from the kinetic data are higher for the carboxymethylated protein than for RNAase A, as are the pKa values of residues 12 and 119. The similar shifts are consistent with the conclusions that both these residues are functionally significant in native and modified enzyme, and that an unblocked tau-nitrogen on histidine-12 is not essential for activity. From the enzyme's catalytic dependence on pH, and the NMR determined pKa values we propose that histidine 12 and 119 function catalytically in their basic and acidic forms respectively.     

Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics and mechanisms of reactions of iron(III) with caffeic acid, chlorogenic acid, sinapic acid, ferulic acid and naringin

The kinetics and mechanisms of the reactions of iron(III) with the hydroxy cinnamic acid based ligands caffeic, chlorogenic, sinapic and ferulic acids and the flavonoid naringin have been investigated in aqueous solution. The mechanisms for caffeic and chlorogenic acid are generally consistent with the formation of a 1:1 complex that subsequently decays through an electron transfer reaction. On reaction with iron(III), ferulic and sinapic acids undergo an electron transfer without the prior formation of any complex. There was no evidence of electron transfer occurring in the complex formed when iron(III) is reacted with naringin. Rate constants for k1 (formation) and k(-1) (dissociation) have been evaluated for the complex formation reactions of [Fe(H2O)6(OH)]2+ with caffeic acid, chlorogenic acid and naringin. Analysis of the kinetic data yielded stability constants, equilibrium constants for protonation of the iron(III) chlorogenic acid complex initially formed, together with the rate constants for complex decomposition through intramolecular electron transfers and in the case of caffeic acid and chlorogenic acid, rate constants for the iron(III) assisted decomposition of the initial complex formed. Some of the suggested mechanisms and calculated rate constants are validated by calculations carried out using global analysis of time dependent spectra.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.