Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-ahphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine. and glutathione have been investigated, with the goal of understanding the relationship between RS^?-Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto ammo acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]_o), pH, and the structure of the redox partners. With excess cysteine present at 25°C, pH 5 0, I = 0 2 M (NaOAc), decay of 1:1 cys-S^?-Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)²⁺ (6 1 × 10³M^?1sec^?1), Cu(Me₅dien)²⁺ (2.7 × 10³M^?1 sec^?1), Cu(en)₂²⁺ (2.1 × 10³M^?1 sec^?1), and Cu(dien)₂²⁺ (4.7 × 10³ M^?1 sec ^?1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results Moderate reactivity decreases in the cysteine-Cu(dien)²⁺, Cu(Me₅dien)²⁺ reactions with increasing [H⁺] (pH 4–6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS^?-Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)²⁺ and Cu(Me₅dien)²⁺. This behavior suggests the mtermediacy of di-μ-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)²⁺ and Cu(Me₅dien)²⁺ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me₆tren)²⁺ (k(25°C) 7.5 × 10⁷ M^?2 sec^?1, I = 0.2 M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR^?-forming pathway. Formation of a coordinated disulfide in the rate-determining step is purposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me₆(tren)Cu(I) product.     

Aquo-pentamine Co(III) complexes as models for carbonic anhydrase

The hydrolysis of 4-nitrophenyl acetate by metal complexes Co(en)2(imH)H2O3+, Co(en)2(bzmH)H2O3+, and Co(en)2(imCH3)H2O3+ (imH = imidazole, bzmH = benzimodazole, imCH3 = methyl imidazole) has been investigated in the pH range 5.4-8.9. The small difference in nucleophilic reactivity in the pH range 5.4-6.7 is assumed to be due to hydrogen bonding abilities of the imidazole and substituted imidazole ligands and small pKa differences (k2(imH) = 2.2 X 10(-2) M-1 sec-1, k2(bzmH) = 5.68 X 10(-2) M-1 sec-1, k2(imCH3) = 1.35 X 10(-2) M-1 sec-1, 40 degrees C, 1 = 0.3 NaClO4, pKa(imH) = 6.2, pKa(imCH3) = 6.2 and pKa(bzmH) = 5.9). In the pH range 7.8-8.9, the differences in nucleophilic reactivity (k3(imH) = 85.5 X 10(-2) M-1 sec-1, k3(bzmH) = 33.4 X 10(-2) M-1 sec-1, 40 degrees C, I = 0.3 NaClO4) are reconciled with a significant steric factor outweighing the acidity of the benzimidazole complex. In the pH region 6.7-7.7, the deviation from linearity is presumably due to both hydroxo and imido ligands functioning as nucleophiles, the latter being about 40 times stronger than the former.     

Kinetics and mechanism of Zn(II) complexation with reduced glutathione

The kinetics of formation and dissociation of mono and bis complexes of Zn(II) with reduced glutathione (H4L+ = fully protonated form) were studied in aqueous solution at 25.0 +/- 0.1 degrees C and ionic strength 0.30 M (NaNO3) in the pH range 4.58 to 4.98 by temperature-jump. The reaction was found to proceed via two different mechanisms depending on degree of ligand protonation. In both cases, complex formation is predominantly if not completely through the sulfur. Reaction with the form HL-2 (only the amino nitrogen protonated), the dominant form of this species, proceeds by the expected rat limiting water loss (dissociative or Eigen) mechanism with rate constants of 9.3 X 10(7) M-1 sec-1 (+/- 24%) for mono and 5.1 X 10(7) M-1 sec-1 (+/- 25%) for bis complex formation. Reaction with H2L--(sulfur protonated) yields rate constants of 3.9 X 10(3) M-1 sec-1 (+/- 43%) for mono and 1.95 X 10(3) M-1 sec-1 (+/- 43%) for bis complex formation. The decrease in rate constant is attributed to blockage of the complexing site on reduced glutathione by intramolecular hydrogen bonding, with proton removal being the rate determining step.     

The reversible reduction of horse metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N,n-tetraacetate.

The reduction of metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetate (FeCDTA2-) has been investigated. The equilibrium constant, measured spectrophotometrically, is 0.21 with a resulting reduction potential of 0.050 V for Mb0. The rate constant for the reduction is 28 M-1 sec-1 with a deltaH ++ of 13 kcal M-1 and deltaS ++ of -11 eu. Both CN- and OH- inhibit the reduction because of the relatively low reactivity of cyanometmyoglobin (Mb+CN-) and ionized metmyglobin (Mb+OH-). The rate constant for the reduction of Mb+CN- by FeCDTA2- is 4.0 X 10(-2) M-1 sec-1 and that for reduction of Mb+OH- is 4.8 M-1 sec-1. The nitric oxide complex of metmyoglobin is reduced with a rate constant of 10 M-1 sec-1. The kinetics of oxidation of oxymyoglobin by FeCDTA- were studied. The data are consistent with a mechanism where oxidation takes place entirely through the deoxy form. A rate constant of 1.45 X 10(2) M-1 sec-1 was calculated for the oxidation of deoxymyoglobin by FeCDTA-, in equilibrium constant and rate constant for reduction. The above data are discussed in terms of a simple outer-sphere reduction reaction.     

Interaction of alpha-lactalbumin with Cu2+

It has been shown by intrinsic fluorescence spectroscopy that alpha-lactalbumin has several Cu2+ -binding sites per molecule. The Ca2+ -loaded protein binds two or more Cu2+ per molecule with an association constant of about 3 X 10(3) M-1. Apo-alpha-lactalbumin binds one Cu2+ per molecule with association constant 8 X 10(4) M-1 and from two to three Cu2+ with an association constant of about 4 X 10(3) M-1. The results obtained from spectrofluorometric pH titration of alpha-lactalbumin in the acidic pH region show the possible involvement of histidine residues in the coordination of Cu2+. The binding of Cu2+ to alpha-lactalbumin lowers significantly its thermostability and stability towards urea denaturation. The stability of Cu2+, Ca2+-alpha-lactalbumin against thermal and urea denaturation is similar to that of the apo protein. The thermal transition in Cu2+, Ca2+-alpha-lactalbumin occurs within the region of physiological temperatures which may suggest the existence of some thermal regulation of its functioning in vivo.     

Catalytic activity of a copper(II)-oxidized glutathione complex on aqueous superoxide ion dismutation

The study of the catalytic activity of a Cu(II)-oxidized glutathione system upon the disproportionation of superoxide radicals shows that the mononuclear complex MA catalyzes dismutation in the pH range 7-9. The corresponding first-order rate constant of value kcat congruent to 6 X 10(6) M-1 sec-1 is pH independent, whereas the second-order rate constant ks for the reference solutions is pH dependent. The kcat constant is about 10-, 100-, and 300-fold higher than the ks constant at pH 7, 8, and 9, respectively. The measured effect is explained in terms of free axial sites in the square-planar arrangement around the copper ion.     

The reaction between ferrous polyaminocarboxylate complexes and hydrogen peroxide: an investigation of the reaction intermediates by stopped flow spectrophotometry

The reactions of Fe(II)EDTA, Fe(II)DTPA, and Fe(II)HEDTA with hydrogen peroxide near neutral pH have been investigated. All these reactions have been assumed to proceed through an active intermediate, I1, (Formula: see text) where pac is one of the three polyaminocarboxylates mentioned above. I1, whether .OH radical or an iron complex, reacts with ethanol, formate, and other scavengers at rates relative to k2 that, with the exception of t-butanol and benzoate, are similar, but not identical, to those expected for the.OH radical. In contrast, at pH 3, in the absence of ligands the reaction of I1 with Fe2+ was inhibited by ethanol and t-butanol and the reactivity of I1 towards these two scavengers relative to ferrous ion is identical to that exhibited by the hydroxyl radical. When pac = HEDTA, the intermediate of the first reaction reacts with formate ion to form the ferrous HEDTA ligand radical complex, which is characterized by absorption maxima at 295 nm (epsilon = 2,640 M-1 cm-1) and 420 nm (epsilon = 620 M-1 cm-1). For the reaction of Fe(II)HEDTA with H2O2, the following mechanism is proposed: (Formula: see text) where k17 = 4.2 X 10(4) M-1 sec-1 and k19 = 5 +/- 0.2 sec-1.     

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.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of bleomycin.     

Interaction of peroxynitrite and hydrogen peroxide with dinitrosyl iron complexes containing thiol ligands in vitro

The interaction of peroxynitrite with thiolate dinitrosyl iron complexes (DNIC) has been examined and compared with the interaction with H2O2. Peroxynitrite oxidized DNIC containing various thiolate ligands--cysteine, glutathione, and bovine serum albumin. Analysis of the oxidation suggested a two-electron reaction and gave third-order rate constants of (9.3 +/- 0.5).109 M-2.sec-1 for DNIC with BSA, (4.0 +/- 0.3).108 M-2.sec-1 for DNIC with cysteine, and (1. 8 +/- 0.3).107 M-2.sec-1 for DNIC with glutathione at 20 degrees C and pH 7.6. Peroxynitrite was more reactive towards DNIC than towards sulfhydryls. Addition of sodium dithionite after the reaction led to significant restoration of the EPR signal of DNIC with cysteine. The reaction of glutathione DNIC with H2O2 was about 600 times slower than with ONOO- and not reversed by sodium dithionite. Thus peroxynitrite, in contrast to hydrogen peroxide, changes the pool of nitrosocompounds which can be responsible for interconversion, storage, and transportation of nitric oxide in vivo.     

Penicillamine deprotonations and interactions with copper ions

In aqueous solutions about five times as many penicillamine molecules possess ionized sulfhydryl groups as deprotonated amino groups. That the corresponding ratio for cysteine is two is reaffirmed. N-Acetyl-dl-penicillamine and d-penicillamine react with Cu(II), even in acid solutions, to yield the corresponding disulfide and Cu(I). Cu(I) binds on the average only one sulfhydryl molecule, and a polymeric structure is suggested. A mixed valence state purple species absorbing at 520 nm and stable even in air is formed when approximately equivalent amounts of Cu(I), Cu(II), and d-penicillamine are present in neutral solutions. In the absence of oxygen and in the presence of 0.1 n base d-penicillamine forms a 2:1 complex with Cu(II) that is stable for hours. Absorption and circular dichroism are reported for the above species and the Cu(II) complexes of l-cystinediamide and l-cystinylbisglycine.     

Hemes and hemoproteins. 3. The reaction of microperoxidase-8 with cyanide: comparison with aquocobalamin and hemoproteins

The monomeric heme octapeptide from cytochrome c, microperoxidase-8, (MP-8), coordinates CN- with log K = 7.55 +/- 0.04 at 25 degrees C in 20% (v/v) aqueous methanol. Log K values are independent of pH between 6 and 9. A spectrophotometric titration of cyanoMP-8 between pH 5.5 and 13.8 gave a single pKa greater than or equal to 13.5 ascribed to ionization of the proximal His ligand. A study of the kinetics of the reaction of MP-8 with cyanide between pH 5.5 and 12, at 25 degrees C and mu = 0.1, indicates that formation of cyanoMP-8 occurs via three routes: attack of CN- on Fe(III) (k1 = 6.0 +/- 0.3 X 10(5) M-1 sec-1); attack of HCN on Fe(III) (k2 = 4.8 +/- 2.0 X 10(3) M-1 sec-1), followed by deprotonation and isomerization to form the C-bound species; and displacement of OH- by CN- when the proximal His ligand is ionized (k5 = 1.8 +/- 0.1 X 10(5) M-1 sec-1). These results are compared with available data for the reaction of cyanide with aquocobalamin and with various hemoproteins.     

Reduction of methemerythrin-anion adducts by dithionite ion

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.     

Oxidation of tryptophan-P-1 and P-2 by beef liver catalase-H2O2 intermediate: comparison with horseradish peroxidase.

Tryptophan-p-1 (trp-p-1) and p-2, which have high mutagenic and carcinogenic potential, are oxidized by catalase- and horseradish peroxidase (HRP)-H2O2 intermediates with optimum pH 5.9 (0.2 M-acetate) in catalase and pH 5.0 (0.2 M-acetate), 8.0 (0.01 M-phosphate) in HRP. The rate constants (k4) of the oxidation in the catalase at pH 5.9 (0.2 M-acetate) were 2965 M-1 x sec-1 for trp-p-1 and 576 M-1 x sec-1 for trp-p-2. In the case of HRP, 1894 M-1 x sec-1, (pH 5.0, 0.2 M-acetate) for trp-p-1 and 705 M-1 x sec-1 (pH 8.0, 0.001 M-phosphate) for trp-p-2 under each optimum condition. The oxidation products of trp-p-1 and p-2 by catalase or HRP lost completely their mutagenic potential in the mutation assay.     

A comparison of the rates of ozonation of biological antioxidants and oleate and linoleate esters

The rates of reaction with ozone of some biological antioxidants and simple polyunsaturated fatty acids (PUFA) have been measured in water or in aqueous micellar solutions. At pH 7.0 the rate constants are ca. 10(6) M-1 sec-1 for urate, alpha-tocopherol, and PUFA, and 6 X 10(7) M-1 sec-1 for ascorbate. When ozone-containing air is breathed, ascorbate in the lung may undergo direct ozonation. However, alpha-tocopherol is probably spared direct reaction with ozone because it doesn't effectively compete with PUFA in pulmonary membranes; rather, tocopherol is used to scavenge radicals produced from the ozone-PUFA reaction.     

Monoclonal antibody to rat apoC: multiple binding to apoC-I on lipoproteins increases its affinity constant

Using solid phase systems, the kinetics of binding of monoclonal antibody (LRB 45, IgG2b,kappa) to apoC-I and apoC-I on lipoproteins were investigated. At 25 degrees C, the association constant of LRB 45 antibody to apoC-I (3.56 X 10(6) M-1 X sec-1) was almost three times slower than the association constant LRB 45 antibody to lipoproteins (10.4 X 10(6) M-1 X sec-1). However, the dissociation constant of apoC-I from LRB 45 antibody (0.865 X 10(-4) sec-1) was also slower than the dissociation constant of lipoprotein from antibody (1.5 X 10(-4) sec-1). Thus, the calculated affinity constant (association constant/dissociation constant) of LRB 45 antibody for apoC-I was approximately half of that for lipoproteins (4.12 X 10(10) M-1 vs. 6.92 X 10(10) M-1). The intrinsic affinity constants for antibody binding to apoC-I and apoC-I on lipoproteins were determined by Scatchard analysis. The intrinsic affinity constant of antibody bound to apoC-I was estimated to be 5.49 X 10(10) M-1 whereas that of antibody binding to lipoproteins was 30 to 200 times less. Furthermore, ascites fluid from LRB 45 cell lines could immunoprecipitate serum lipoproteins. Thus, it is concluded that there is multiple binding of antibody to apoC-I on lipoproteins. This binding appears to increase the calculated affinity constant (avidity) for antibody-antigen interaction.     

Interaction of some Pd(II) complexes with Na+ / K+-ATPase: inhibition, kinetics, prevention and recovery

The aim of this work was to investigate the influence of [PdCl4]2-, [PdCl(dien)]+ and [PdCl(Me4dien)]+ complexes on Na+ / K+-ATPase activity. The dose-dependent inhibition curves were obtained in all cases. IC50 values determined by Hill analysis were 2.25 x 10(-5) M, 1.21 x 10(-4) M and 2.36 x 10(-4) M, respectively. Na+ / K+-ATPase exhibited typical Michelis-Menten kinetics in the presence of Pd(II) complexes. Kinetic parameters (Vmax, Km) derived using Eadie-Hofstee transformation indicated a noncompetitive type of Na+ / K+-ATPase inhibition. The inhibitor constants (Ki) were determined from Dixon plots. The order of complex affinity for binding with Na+ / K+-ATPase, deducted from Ki values, was [PdCl4]2- > [PdCl(dien)]+ > [PdCl(Me4dien)]+. The results indicated that the potency of Pd(II) complexes to inhibit Na+/ K +-ATPase activity depended strongly on ligands of the related compound. Furthermore, the ability of SH-donor ligands, L-cysteine and glutathione, to prevent and recover the Pd(II) complexes-induced inhibition of Na+ / K+-ATPase was examined. The addition of 1 mM L-cysteine or glutathione to the reaction mixture before exposure to Pd(II) complexes prevented the inhibition by increasing the IC50 values by one order of magnitude. Moreover, the inhibited enzymatic activity was recovered by addition of SH-donor ligands in a concentration-dependent manner.     

Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.     

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.