Quantitative characteristics of interactions of blood lipoproteins and steroid hormones]

Human and serum lipoproteins interaction with steroid hormones (corticosterone and hydrocortisone) were studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoids binding ability. Association constants were found to be 0.6-2.0 x 10 M for corticosterone and 4.0-8.0 x 10 M for hydrocortisone. The number of binding sites ranged from 3 to 300 for different classes of lipoproteins. Our data suggest high specificity of serum lipoproteins binding with corticosterone and hydrocortisone.     

Binding affinity and reactivity of lecithin cholesterol acyltransferase with native lipoproteins.

The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a reconstituted discoidal HDL (rHDL) were determined by the activity-inhibition method. In addition, enzyme kinetics were measured with each of the lipoprotein substrates. Based on phospholipid concentrations, the Kd values (0.9 x 10(-5) to 4.6 x 10(-5) M) increased in the order rHDL = HDL3 相似文献   

Interaction of lipoproteins in blood serum with steroid hormones

Interaction of human and serum lipoproteins with steroid hormones (corticosterone and cortisol) was studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoid-binding ability. Association constants were found to be 0.6-2.0 x 10(6) M-1 for corticosterone and 4.0-8.0 x 10(6) M-1 for cortisol. The number of binding sites varied from 3 to 300 for different classes of lipoproteins.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.     

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. II. The Ca2+-arsenazo III complexes

The analytical method described in the preceding article was applied to spectrophotometric Ca2+-titrations of the metallochromic indicator arsenazo III (Ar). At various reactant concentrations it was determined that Ar forms 1:1,1:2 and 2 : 1 complexes with calcium. The equilibrium constants and extinction coefficients at 602 nm were determined. Corrected to zero ionic strength at 293 K and pH 7.0, the reactions Ca + Ar = CaAr, CaAr + Ar = CaAr2 and CaAr + Ca = Ca2Ar are associated with dissociation equilibrium constants k(11) = 1.6 x 10(-6)M, K12 = 3.2 x 10(-4)M and K21 = 5.8 x 10(-3)M. respectively. The extinction coefficient of unbound indicator is (602) = 9.6 (+/-0.3) x 10(3) cm(-1) M(-1). Arscnazo III complexes with monovalent ions like Na+ and K+ : at zero ionic strength, the dissociation constant of the Na+-Ar complex is about 0.1 M.     

Protease nexin II interactions with coagulation factor XIa are contained within the Kunitz protease inhibitor domain of protease nexin II and the factor XIa catalytic domain

Protease nexin II, a platelet-secreted protein containing a Kunitz-type domain, is a potent inhibitor of factor XIa with an inhibition constant of 250-400 pM. The present study examined the protein interactions responsible for this inhibition. The isolated catalytic domain of factor XIa is inhibited by protease nexin II with an inhibition constant of 437 +/- 62 pM, compared to 229 +/- 40 pM for the intact protein. Factor XIa is inhibited by a recombinant Kunitz domain with an inhibition constant of 344 +/- 37 pM versus 422 +/- 33 pM for the catalytic domain. Kinetic rate constants were determined by progress curve analysis. The association rate constants for inhibition of factor XIa by protease nexin II [(3.35 +/- 0.35) x 10(6) M(-1) s(-1)] and catalytic domain [(2.27 +/- 0. 25) x 10(6) M(-1) s(-1)] are nearly identical. The dissociation rate constants are very similar, (9.17 +/- 0.71) x 10(-4) and (7.97 +/- 1.1) x 10(-4) s(-1), respectively. The rate constants for factor XIa and catalytic domain inhibition by recombinant Kunitz domain are also very similar: association constants of (3.19 +/- 0.29) x 10(6) and (3.25 +/- 0.44) x 10(6) M(-1) s(-1), respectively; dissociation constants of (10.73 +/- 0.84) x 10(-4) and (10.36 +/- 1.3) x 10(-4) s(-1). The inhibition constant (K(i)) values calculated from these kinetic parameters are in close agreement with those measured from equilibrium binding experiments. These results suggest that the major interactions required for factor XIa inhibition by protease nexin II are localized to the catalytic domain of factor XIa and the Kunitz domain of protease nexin II.     

Kinetics and thermodynamics of lipid amphiphile exchange between lipoproteins and albumin in serum

We have examined the kinetics and thermodynamics of the exchange of a fluorescent amphiphile derived from a phospholipid, NBD-DMPE, between serum albumin and the serum lipoproteins of high density (HDL2 and HDL3), LDL, and VLDL. Binding of the fluorescent lipid amphiphile to bovine serum albumin is characterized, at 35 degrees C, by an equilibrium binding constant of approximately 3 x 10(6) M(-1) and a characteristic time < or = 0.1 s. Association of NBD-DMPE with the lipoprotein particles, if considered as a partitioning of amphiphile monomers between the aqueous phase and the lipoprotein particles, is characterized by an equilibrium partition coefficient between 10(5) and 10(6), being highest for LDL and lowest for HDL. The association of NBD-DMPE monomers with lipoprotein particles can be described by insertion rate constants on the order of 10(5) M(-1) s(-1) for VLDL and LDL and 10(4) M(-1) s(-1) for HDL. The desorption rate constants are on the order of 10(-5) s(-1) for all particles. The study was performed as a function of temperature between 15 and 35 degrees C. This permitted the calculation of the equilibrium thermodynamic parameters (deltaG(o), deltaH(o), and deltaS(o)) as well as the activation parameters (deltaG++(o), deltaH++(o), and deltaS++(o)) for the insertion and desorption processes. The association equilibrium is dominated by the entropic contribution to the free energy in all cases. The results are discussed in relation to phospholipid and amphiphile exchange phenomena involving the lipoproteins.     

Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Study on the interactions between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 x 10(3)M(-1) for active compound C(3) and 8.42 x 10(3)M(-1) for MC(3) at 20.0 degrees C, 0.626 x 10(3)M(-1) and 0.644 x 10(3)M(-1) at 37.0 degrees C, respectively. The thermodynamic parameters Delta H and DeltaS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.     

Ganglioside GM3 stimulates the uptake and processing of low density lipoproteins by macrophages

Preincubation of low density lipoproteins (LDL) with low concentrations of the ganglioside GM3 (1-2x 10(-5) M/2.5 x 10(-6) M LDL-protein) results in an increase of LDL-uptake, enhances cholesterol accumulation and cholesteryl ester formation by macrophages. At the same time the lysosomal degradation of LDL in macrophages was inhibited under these conditions. These effects depended on the ganglioside structure and concentration. It is suggested that the effects observed could be caused by GM3-induced modification of LDL to a form that becomes recognized by macrophages.     

Kinetic analysis of acetylcholine-induced chloride current in isolated snail neurons

1. Kinetics of activation and desensitization phases of the acetylcholine (ACh)-induced chloride current (ICI) were studied using isolated single neurons of Japanese land snail and the "concentration clamp" technique. 2. The dose-response curve for the peak ICI gave a dissociation constant of 7.1 x 10(-6) M and a Hill coefficient of 1.8. 3. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV) and the reversal potential (EACh) was -7.2 +/- 1.5 mV (N = 10). The value was close to the calculated equilibrium potential for chloride ions (ECI). 4. Both activation and desensitization phases of the ACh-induced ICI consisted of a single exponential at concentrations less than 3 x 10(-6) M and a double exponential at higher concentrations. The time constants of both phases decreased with increasing ACh concentrations but showed no potential dependency. 5. The recovery from desensitization of the ICI induced by 5 x 10(-6) M ACh proceeded double exponentially, with time constants of 11 and 114 sec at a holding potential of -30 mV. 6. Noise analysis was performed on a steady-state current induced by 3 x 10(-7) to 2 x 10(-6) M ACh. The mean open time was about 60 msec at 10(-6) M ACh and the single-channel conductance was 14 PS. 7. These results suggest that the ACh receptor-Cl channel complex in snail neurons has two binding sites with the dissociation constant of 7.1 x 10(-6) M and is rapidly activated and desensitized to a steady level in the presence of the agonist.     

Kinetic and mechanistic studies of the NO*-mediated oxidation of oxymyoglobin and oxyhemoglobin

The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy, increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10(6) M(-1) x s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M(-1) x s(-1) for oxyhemoglobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M(-1) x s(-1) and (144 +/- 3) x 10(6) M(-1) x s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO* depend neither on the association grade of the protein (dimer/tetramer) nor on the concentration of the phosphate buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyoglobin and oxyhemoglobin proceed via intermediate peroxynitrito complexes which were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorption maxima around 500 and 635 nm. These species can be observed at alkaline pH but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing pH and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these reactions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting the proteins with NO* for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without nitrating the globins in physiologically significant amounts.     

Interaction of metal ions with caffeine and theophylline: stability and structural features

The interactions of caffeine and theophylline with divalent cadmium, mercury, strontium and barium ions were studied in aqueous solution and physiological pH. Fourier transform infrared spectroscopy (FTIR) and absorption spectra were used to determine the cation binding mode and association constants. Spectroscopic results showed that Cd(2+), Hg(2+), Sr(2+) and Ba(2+) bind strongly to caffeine and theophylline. Direct and indirect (through metal hydration shell) interactions were observed for caffeine and theophylline with Cd(2+), Hg(2+), Sr(2+) and Ba(2+) through O6 and N9 (caffeine) and O6, N9 and N7 atoms (theophylline). The overall binding constants are:k(Cd-caffeine) = 1.24 x 10(5) M(-1), k(Hg-caffeine) = 1.74 x 10(5) M(-1), k(Sr- caffeine) = 3.3 x 10(4) M(-1), k(Ba-caffeine) = 1.8 x 10(4) M(-1), k(Cd-theophylline) = 5.75 x 10(5) M(-1), k(Hg-theophylline) = 2.14 x 10(5) M(-1), k(Sr-theophylline) = 4.6 x 10(4) M(-1), k(Ba-theophylline) = 3 x 10(4) M(-1). These k values are evidence for weak and strong cation interactions in these metal complexes.     

Hemoglobin equilibrium analysis by the multiangle laser light-scattering method

Dimer-tetramer and monomer-dimer-tetramer equilibria of tetrameric hemoglobins and their single chains in the CO form, respectively, were evaluated using the microbatch multiangle light-scattering (MALS) analysis system. The molecular weights of human Hb A and Hb F in the CO form were dependent on concentration. The dissociation constants to dimers of Hb A and Hb F were 2.58 x 10(-6) and 0.66 x 10(-6), respectively. Equilibration of single globin chains, including alpha, beta, and gamma chains, was also evaluated by the same method. The dissociation constants of alpha-chain dimers to monomers, of beta-chain tetramers to monomers, and of gamma-chain tetramers to dimers were 14 x 10(-6), 25 x 10(-17), and 6.86 x 10(-6) M, respectively. These results indicate that the MALS analysis system can not only determine molecular weight but also characterize protein-protein interactions of multi-subunit proteins.     

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

The kinetics and mechanisms of the complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III)

(-)-Epigallocatechin-gallate ((-)-EGCg) and (-)-epicatechin-gallate ((-)-ECG) are important antioxidants which are found in green tea. The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with EGCg and ECG have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are consistent with a mechanism in which the 2:1 metal:ligand complex initially formed on reaction of iron(III) with the ligand subsequently decomposes in an electron transfer step. Complex formation takes place at two separate binding sites via coupled reactions. Rate constants of 4.28(+/-0.06) x 10(6) M(-2) s(-1) and 2.83(+/-0.04) x 10(6) M(-2) s(-1) have been evaluated for the reaction of monohydroxy Fe(OH)2+ species with EGCg and ECG, respectively while rate constants for of 2.94(+/-0.4) x 10(4) M(-2) s(-1) and 2.41(+/-0.25) x 10(4) M(-2) s(-1) have been evaluated for the reaction of Fe3+ species with EGCg and ECG, respectively. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with EGCg and ECG were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra. The results obtained show that one molecule of either EGCg or EGC is capable of reducing up to four iron(III) species, a fact which is consistent with the powerful antioxidant properties of the ligands.