Singlet molecular oxygen [O<Subscript>2</Subscript>(<Superscript>1</Superscript>Δ<Subscript>g</Subscript>)]-mediated photodegradation of tyrosine derivatives in the presence of cationic and neutral micellar systems

The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC相似文献   

Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives

The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage.     

Micellar effect on the scavenging of singlet molecular oxygen by hydroxybenzenes

The antioxidative effectiveness of three hydroxyaromatic derivatives (OHAD), namely phenol, resorcinol and phloroglucinol, as reflected by their scavenging ability of the photochemically generated species singlet molecular oxygen [O(2)((1)Delta(g))], was studied in aqueous solution and micellar media. Kinetic results, obtained through time-resolved phosphorescence detection of O(2)((1)Delta(g)) emission and polarographic methods, at pH 7 and 12 in homogeneous and CTAB micellar media, were compared. Calculated photooxidation quantum efficiencies (phi(r)), ranging from very low values ( approximately 0.01) to relatively high ones (approximately 0.3), depend on the preferential solubilization sites of the hydroxyaromatic derivatives either in the micellar or homogeneous media. OHAD in water pH 7 and in CTAB 0.02 M pH 12 exhibit their highest antioxidative efficiency as O(2)((1)Delta(g)) scavengers. In these conditions, they present the best degree of self protection against O(2)((1)Delta(g))-mediated photooxidation with relatively high values for the overall quenching rate constant and, simultaneously, the lowest phi(r).     

The balance between charge transfer and non-charge transfer pathways in the sensitization of singlet oxygen by pi pi* triplet states.

A charge transfer (CT) channel and a non-CT deactivation channel, both leading to formation of O(2)((1)Sigma (g)(+)), O(2)((1) Delta(g)) and O(2)((3)Sigma(g)(-)), compete in the quenching of triplet states by O(2). Recent studies by our group demonstrated that these channels are described by rather simple and general quantitative relations. In the present paper we use the detailed kinetic data on the quenching by O(2) of pi pi* triplet sensitizers of three homologous aromatic series in CCl(4) to derive a parameter, which describes the balance between CT and non-CT deactivation. This quantity, p(CT), is the relative contribution of CT mediated deactivation and is easily calculated for a sensitizer of known triplet energy from its quenching rate constant. The parameter p(CT) quantitatively describes the balance between both deactivation channels without requiring any knowledge of oxidation potentials. It is shown how the variation of p(CT) influences the efficiencies and the rate constants of O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)) and O(2)((3)Sigma(g)(-)) formation in the quenching process.     

The role of vitamin B6 as an antioxidant in the presence of vitamin B2-photogenerated reactive oxygen species. A kinetic and mechanistic study

We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Vitamin B2-sensitised photooxidation of the ophthalmic drugs Timolol and Pindolol: kinetics and mechanism

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with catechins (catechin (CA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG)) and related compounds (5-methoxyresorcinol (MR), 4-methylcatechol (MC), and n-propyl gallate (PG)) was performed in ethanol at 35 degrees C. MR, MC, and PG are considered to be a model of resorcinol (A)-, catechol (B)-, and gallate (G)-rings in catechins, respectively. The overall rate constants, k(Q) (= k(q) + k(r), physical quenching + chemical reaction), for the reaction of catechins with (1)O(2) increased in the order of PG < MR < MC < CA < EC < EGC < ECG < EGCG. In a comparison of the rate constants, the relationship between quenching rates and chemical structures is discussed. The catechins which have lower peak oxidation potentials, E(P), show higher reactivities. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by catechins. The k(Q) values of EGCG (1.47 x 10(8) M(-1) s(-1)) and ECG (7.81 x 10(7)) were found to be larger than those of lipids (1.3 x 10(5)-1.9 x 10(5) M(-1) s(-1)), amino acids (<3.7 x 10(7)), and DNA (5.1 x 10(5)). Further, these values are similar to those (1.15 x 10(8)-2.06 x 10(8) M(-1) s(-1)) of alpha- and gamma-tocopherol, ubiquinol-10, and gamma-tocopherol hydroquinone (plastoquinol model). The result suggests that catechins may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Reactions of e(-)(aq), CO(2)(*)(-), HO(*), O(2)(*)(-) and O(2)((1)delta(g)) with a dendro[60]fullerene and C(60)[C(COOH)(2)](n) (n = 2-6)

Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process.     

Effect of reverse micelles on the Rose Bengal-sensitized photo-oxidation of 1- and 2-hydroxynaphthalenes

Benzylhexadecyl dimethylammonium chloride (BHDC) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) water-in-oil micro-emulsions were employed to study the influence of medium heterogeneity on singlet molecular oxygen [O(2)((1)Delta(g))]-mediated degradation of 1-hydroxynaphthalene (1-OHN) and 2-hydroxynaphthalene (2-OHN) in their naphthoate form. The kinetic study in the micellar system, that was considered as a closer model for the environment of contaminants in natural polluted waters, consisted of a comparative work with the process in homogeneous solution, by varying surfactant structure and water content of the micro-emulsion. While it is known that 1-OHN and 2-OHN are rapidly and efficiently photo-oxidised in aqueous medium, time-resolved phosphorescence detection of O(2)((1)Delta(g)) and stationary photolysis experiments demonstrate that both the values for the overall and reactive rate constants for the quenching of O(2)((1)Delta(g)) and the photo-oxidation efficiencies are lowered in BHDC micelles, whereas the photo-oxidative process in AOT micro-emulsions was totally inhibited. Results are interpreted and discussed on the basis of different locations of the probe in the micellar environment.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B2 (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (3Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of 3Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O2*-) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O2(1delta(g)), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O2(*-)-mediated mechanism. BHA mainly interacts with O2(1delta(g)) and exhibits a desirable property as an antioxidant--a relatively high capacity for O2(1delta(g)) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Electronic effects in the acylation of alpha-chymotrypsin by substituted N-benzoylimidazoles

Rate constants for the acylation of alpha-chymotrypsin by a series of acyl-substituted N-benzoylimidazoles have been determined by proflavin displacement from the active site. The second-order acylation rate constants k2/Km are large [e.g., that for N-(m-nitrobenzoyl)imidazole is 1.7 X 10(4) M-1 s-1 at pH 7.5], even though Km must be quite large (plots of k vs. k/[S]0 have infinite slopes). The values of k2/Km are nearly independent of pH in the range 5.0-9.0 when the substituent group is electron donating. Electron-withdrawing substituents produce an increase in k2/Km with increasing pH until a maximum is reached near pH 7. This is also the case in acylation by the N-[p-(dimethylamino)benzoyl]-N'-methylimidazolium ion (pKapp = 6.5). While the reaction of the N'-methylated derivative is via a positively charged species at all pH values, the unmethylated compounds react through both the neutral species and the conjugate acids, with the observed pH dependence depending on the relative values of the rate constants. The limiting value of k2/Km for the N-[p-(dimethylamino)benzoyl]-N'-methylimidazolium ion is 2.1 times less in D2O than in H2O. Thus, His-57 must be participating in the acylation reaction as a general base. The limiting values of k2/Km for the corresponding N'-methylated and unmethylated derivatives differ by a factor of only 150, which is similar to the difference in the second-order rate constants for nonenzymatic OH- -catalyzed hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.     

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.     

Interconversion of Cu(I) and Cu(II) forms of galactose oxidase: comparison of reduction potentials

A procedure for the preparation of the fully reduced Cu(I) form of galactose oxidase, GOase(red), involving reduction of GOase(semi) (or GOase(ox)) with non-coordinating [Ru(NH(3))(6)](2+) (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH(3))(6)](2+) give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOase(semi) (k(f)=860 M(-1) s(-1)) give a first-order [H(+)]-dependence (pK(1a)=7.9), but the reverse process involving [Ru(NH(3))(6)](3+) oxidation of GOase(red) (k(b)=18.6 M(-1) s(-1)) is independent of pH (5.5 to 9.5). The reduction potential E(2)(o)' (vs. nhe) for the GOase(semi)/GOase(red) (i.e. Cu(II)/Cu(I)) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK(1a) (GOase(semi)) and pK(2a) (GOase(red)) acid dissociation constants both involving Tyr-495. It is concluded that pK(2a) is for acid dissociation of uncoordinated H(+)Tyr-495. Consistent with this interpretation rate constants/M(-1) s(-1) for the GOase(semi) Tyr495 Phe variant, k(f)=1.59x10(3) and k(b)=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E(1)(o)'/mV pH 7.5) for the GOase(ox)/GOase(semi) (i.e. Tyr(.)/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N(3)(-) for H(2)O at the substrate binding exogenous site (393), and for apo-protein (570). These compare with previously reported values for the variants Trp290His (730) and Tyr495Phe (450), and together serve to quantify different contributions to the unusually small E(1)(o)' of 400 mV for the Tyr(.)/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOase(ox)/GOase(red) couple is calculated to be 275 mV. The rate constant for the reaction of GOase(red) with GOase(ox) is 4.4x10(3) M(-1) s(-1) at pH 7.5.     

Structure-activity relationship of the tocopherol-regeneration reaction by catechins

The reaction rates (k(r)) of 5,7-diisopropyl-tocopheroxyl radical (Toc) with catechins (epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG)) and related compounds (methyl gallate (MG), 4-methylcatechol (MC), and 5-methoxyresorcinol (MR)) have been measured by stopped-flow spectrophotometer. The k(r) values increased in the order of MR < < MG < EC < MC approximately ECG < EGC < EGCG in ethanol and 2-propanol/H(2)O (5/1, v/v) solutions, indicating that the reactivity of the OH groups in catechins increased in the order of resorcinol A-ring < < gallate G-ring < catechol B-ring < pyrogallol B-ring. The catechins which have lower oxidation potentials show higher reactivities. The rate constants for catechins in micellar solution showed notable pH dependence with one or two peaks around pH 9-11, because of the dissociation of various phenolic hydroxyl protons in catechins. The structure-activity relationship in the free-radical-scavenging reaction by catechins has been clarified by the detailed analyses of the pH dependence of k(r) values. The reaction rates increased remarkably with increasing the anionic character of catechins, that is, the electron-donating capacity of catechins. The mono anion form at catechol B-and resorcinol A-rings and dianion form at pyrogallol B-and gallate G-rings show the highest activity for free-radical-scavenging. It was found that catechins (EC, ECG, EGC, and EGCG) have activity similar to or higher than that of vitamin C in vitamin E regeneration at pH 7-12 in micellar solution.     

Speciation of phytate ion in aqueous solution. Sequestration of magnesium and calcium by phytate at different temperatures and ionic strengths, in NaCl(aq)

The formation and stability of Mg(2+) and Ca(2+)-phytate complexes was studied potentiometrically using an ISE-H(+) electrode. Measurements were performed at 10 degrees C and 25 degrees C in NaCl(aq) in the ionic strength range 0.1< or =I< or =0.75 mol L(-1). For both magnesium and calcium systems, the formation of ten M(i)PhyH(j)((12-2i-j)-) species was observed in the range 3< or =pH< or =7 with i=1, 2, 3 and j=3, 4, 5 (and i=3, j=2). These species are quite stable; here we report for example some quantitative data for the species Ca(i)PhyH(3)((9-2i)-), i=1, 2, 3 (equilibrium iCa(2+)+H(j)Phy((12-j)-)=Ca(i)PhyH(j)((12-j-2i)-): K(ij)) at I=0.25 mol L(-1) and t=25 degrees C: logK(13)=3.42, logK(23)=6.47 and logK(33)=9.41. The speciation of the Ca(2+)-phytate system was also checked by ISE-Ca(2+) measurements. Dependence on ionic strength was modeled using a simple Debye-Hückel type equation and formation constants were calculated at infinite dilution. The stability constants of complexes formed at pH>7 were estimated using an empirical predictive equation. The sequestering ability of phytate towards Mg(2+) and Ca(2+) was calculated in different experimental conditions and compared with those of other chelating agents.