Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Oxidation of Manganous and Ferrous Metalloporphyrins by Dioxygen in Aqueous Solutions

The stoichiometry and rate of oxidation with dioxygen of tetra-(p-sulfonatophenyl)-porphinatomanganese(II) and the bisimidazole tetra(p-sulfonatophenyl)porphinato-iron(II) were studied in aqueous solutions at neutral pH. The stoichiometry for both complexes was determined; two molecules of metalloporphyrin reacted with dioxygen to produce the +3 oxidation state of the metalloporphyrins and hydrogen peroxide. The rate law for the oxidation of Mn(II)-TPPS is rate = k′[Mn(II)-TPPS][O₂], with k′ at 26.5° of 2.6 × 10⁵ M^?1 sec^?1. The rate law for the oxidation of Fe(II)-TPPS in the presence of imidazole is

with k″ = 10,100 sec^?1. Some possible mechanisms consistent with these data are discussed.     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

Reaction of imidazole and hydroquinone with oxymyoglobin

Oxymyoglobin reacts with imidazole, substituted imidazoles, and hydroquinone to give metmyoglobin. The kinetics of these reactions have been studied. The rates are first order in both reactants, and second-order rate constants are reported. At pH 8.2, k₁ for imidazole is 2.5 ± 0.3 × 10^?3 M^?1 sec^?1 and for hydroquinone is 4 ± 0.4 × 10^?1 M^?1 sec^?1. The rates are independent of pH for imidazole but increase rapidly with pH for hydroquinone. The mechanism for all these reactions is thought to involve the two-electron reduction of molecular oxygen to peroxide with concurrent oxidation of both the protein and the reactant. An analogous mechanism has been suggested previously [1] for the reaction of oxyhemoglobin with hydroquinone. It has previously been shown [6] that imidazole can mediate the transfer of electrons to heme proteins by forming a transient reduced radical. The present results indicate that it can also form a transient oxidized radical under mild conditions. This dual capability may be important in biological electron-transfer processes.     

Kinetics of reduction of ferrioxamine B by chromium(II), vanadium(II), and dithionite

Kinetic studies of the reduction of ferrioxamine B (Fe(Hdesf)⁺) by Cr(H₂O)₆²⁺, V(H₂O)₆²⁺, and dithionite have been performed. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the rate is ?d[Fe(Hdesf)⁺]/dt = k[Fe(Hdesf)⁺][M²⁺]. For Cr(H₂O)₆²⁺, k = 1.19 × 10⁴ M^?1 sec^?1 at 25°C and μ = 0.4 M, and k is independent of pH from 2.6 to 3.5. For V(H₂O)₆²⁺, k = 6.30 × 10² M^?1 sec^?1 at 25°C, μ = 1.0 M, and pH = 2.2. The rate is nearly independent of pH from 2.2 to 4.0. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the activation parameters are ΔH^≠ = 8.2 kcal mol^?1, ΔS^≠ ?12 eu and ΔH^≠ = 1.7 kcal mol^?1, ΔS^≠ = ?40 eu (at pH 2.2) respectively. Reduction by Cr(H₂O)₆²⁺ is inner-sphere, while reduction by V(H₂O)₆²⁺ is outer-sphere. Reduction by dithionite follows the rate law ?d[Fe(Hdesf)⁺]/dt =kK^{$\text{1}\text{2}$}[Fe(Hdesf)⁺][S₂O₄^2?]^{$\text{1}\text{2}$} where K is the equilibrium constant for dissociation of S₂O₄^2? into SO₂^? radicals. The value of k at 25°C and μ = 0.5 is 2.7 × 10³ M^?1 sec^?1 at pH 5.8, 3.5 × 10³ M^?1 sec^?1 at pH 6.8, and 4.6 × 10³ M^?1 sec^?1 at pH 7.8, and ΔH^≠ = 6.8 kcal mol^?1 and ΔS^≠ = ?19 eu at pH 7.8.     

Binding kinetics of sulfatide with influenza A virus hemagglutinin

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k _on) of 1.41?×?10⁴ M^?1 sec^?1, dissociation rate constant (k _off) of 2.03?×?10^?4 sec^?1 and K _d of 1.44?×?10^?8 M to sulfatide immobilized on a sensor chip. The k _off values of sHA were similar for sulfatide and GD1a, whereas the k _on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.     

Reactions of iron-sulfur proteins with the aquated electron

The reaction of parsley 2Fe-2S ferredoxin in the normal oxidized state with e_aq^? generated by pulse radiolysis techniques has been studied at ～25°C, pH 7–8, I = 0.10 M (NaClO₄). Rate constants k_e (e_aq^? decay) and k_p (protein absorbance change) are the same, second-order rate constant 9.7 × 10⁹ M^?1 sec^?1. The reaction exhibits close to 100% efficiency. With 8Fe-8S ferredoxin from Clostridium pasteurianum under identical conditions it now appears that k_p (although sometimes significantly smaller) is equal to k_e. Varying efficiencies are also observed with this protein depending on the batch used. The reasons for such variable behavior are not fully understood. With oxidized and reduced forms of Chromatium v. high-potential iron-sulfur protein (HIPIP), k_e and k_p are essentially the same, but the highest efficiency observed is only ～50%. The prevailing pattern is therefore that rate constants k_e and k_p are generally in step for proteins having a single (or identical) active site(s). When the active site is buried as with HIPIP the efficiency of the reaction appears to decrease.     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

Self-exchange rates and electron transfer kinetics of horse heart ferricytochrome C with amino acid-pentacyanoferrate(II) complexes

The electron transfer reactions of horse heart cytochrome c with a series of amino acid-pentacyanoferrate(II) complexes have been studied by the stopped-flow technique, at 25°C, μ = 0.100, pH 7 (phosphate buffer). A second-order behavior was observed in the case of the Fe(CN)₅ (histidine)^3? complex, with k = 2.8 x 10⁵ M^?1 sec^?1. For the Fe(CN)₅ (alanine)^4? and Fe(CN)₅(L-glutamate)^5? complexes, only a minor deviation of the second-order behavior, close to the experimental error (k = 3.2 × 10⁵ and 1.6 x 10⁵ M^?1 sec^?1, respectively) was noted at high concentrations of the reactants (e.g., 6 × 10^?4 M). The results are in accord with recent work on the Fe(CN)₆^4?/cytochrome c system demonstrating weak association of the reactants. The calculated self-exchange rate constants including electrostatic interactions for the imidazole,L -histidine, 4-aminopyridine, glycinate, β-alaninate, andL-glutamate pentacyanoferrate(II) complexes were 3.3 × 105, 3.3 × 10⁵, 2.8 × 10⁶,4.1 × 10²,5.5 × 10², and 6.0 M^?1 sec^?1, respectively. Marcus theory calculations for the cytochrome c reactions were interpreted in terms of two nonequivalent binding sites for the complexes, with the metalloprotein self-exchange rate constants varying from 10⁴ M^?1 sec^?1 (histidine, imidazole, and 4-aminopyridine complexes) to 10⁶ M^?1 sec ^?1 (glycinate, β-alaninate, and L-glutamate complexes).     

Pulse radiolytically generated superoxide and Cu(II)-salicylates.

The rate constants of the reactions between pulse radiolytically produced superoxide anions and the Cu(II) chelates of salicylate, acetylsalicylate, p-aminosalicylate and diisopropylsalicylate were determined at pH 7.5 and found to range from 0.8 to 2.4 × 10⁹ M^?1 sec^?1. It was intriguing to note that they had a superoxide dismutase activity identical with that of native cuprein-copper (k₂₄₅ = 1.3 × 10⁹ M^?1 sec^?1 per g-atom of Cu). These measurements confirm our earlier observations using indirect assays that all copper salicylates act as perfect model superoxide dismutases and favour the proposal that the activity of anti-inflammatory agents might be assigned to their in vivo formed Cu complexes.     

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK～ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (k_cat/K_M = 6.83 × 10⁶ M^?1 sec^?1) and DDDDR (k_cat/K_M = 1.89 × 10⁷ M^?1 sec^?1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge–charge interactions in the extended substrate binding pocket.     

Catalysis by polymer complexes. I. Enhanced esterolytic reactivity of hydroxamate–polymer complexes

N-methylmyristohydroxamic acid (1) bound to polymer micelles of laurylated poly(2- and 4-vinylpyridines) (lauryl group contet: 2VP-L, 30 mol%; 4VP-L, 33 mol%) quantitatively reacted with p-nitrophenyl acetate (NpAc) within a few seconds at 30°C, pH 8.95. Second order rate constants k_a were 34,000 M^?1 sec^?1 for 1–2VP-L and 11,400 M^?1 sec^?1 for 1–4VP-L at μ = 0.5, and they were pronouncedly improved by a decrease in ionic strength (k_a = 27,500–80,200 M^?1 sec^?1 at μ = 0.08). In contrast, poly(N-ethyl-4-vinylpyridinium bromide) hardly affected the nucleophilicity of the hydroxamate ion. Therefore, the enhancement was considered to be associated with some micellar characteristics. Typical saturation phenomena of the reaction rate were observed for p-nitrophenyl hexanoate (NpOCOPe) and 3-nitro-4-acetoxybenzoic acid (NpAcCOOH). It was suggested that binding of NpOCOPe is caused by the hydrophobic interaction, while that of NpAcCOOH is probably induced by the electrostatic interaction. It is demonstrated that the cationic polymer micelle enormously activates the bound hydroxamate anion, and these complexes would be of much interest as a biomimetic system for enzyme catalysis.     

Kinetics of some electron-transfer reactions in biological photosystems. I. Pulse radiolysis study of spinach ferredoxin reduction by the hydrated electron and CO−2 radical

The reduction of spinach ferredoxin by the CO^?₂ radical and the hydrated electron (e^?_aq) has been studied by pulse radiolysis in the pH range between 5.05 and 9.67. The reduction of oxidized spinach ferredoxin by both CO^?₂ and e^?_aq was found to be essentially quantitative. The CO^?₂ radical reduces spinach ferredoxin by a single second-order process at a rate k₅ = (6.2 ± 0.6) · 10⁷ M^?1 · s^?1. Reduction by e^?_aq follows a biphasic pathway. The first phase obeys second-order kinetics for the reduction of the cluster, k_app = (9.4 ± 0.3) · 10⁹ M^?1 · s^?1. The second phase follows an intramolecular first-order reaction k_B = (8.3 ± 1.7) · 10² s^?1 which is observed as a further reduction of the active site. Spectral changes accompanying the reduction of oxidized spinach ferredoxin in the ultraviolet and visible range are discussed.     

Nitrofuran induced mutagenesis and error prone repair in Escherichia coli

The binding of cis(c)- and trans(t)-Pt(NH₃)₂Cl₂ to DNA at platinum/DNA-nucleotide ratios (R_i) of 0.1 or less has been studied by means of radioactive ^195mPt-labeled compounds. Kinetic data are consistent with the following scheme:

At 25°C and pH 5–6 in 5 mM NaClO₄, the values for the rate constants in the above scheme for the c-isomer are k₂ = 2.2 × 10^?5 sec^?1, k₇ = 0.32 (sec M)^?1, and k₈ = 143 (sec M)^?1; for the t-isomer the values are k₂ < 0.5 × 10^?5 sec^?1 and k₇ = 0.95 (sec M)^?1. Platinum-DNA adducts do not undergo detectable exchange after 3 days at 37°C, indicating the absence of a dynamic equillibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag⁺ and H⁺ titration studies are consistent with binding at guanine N(7) for R_i < 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37°C and pH 7 in 0.15 M NaCl, 6–8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.     

Kinetics and mechanism of catalase action. Formation of the intermediate complex

The kinetics of formation of the intermediate complex between catalase and H₂O₂ has been reexamined. It has been shown that the kinetics consists of a rapid and of a subsequent slow phase. At the maximum of the transient decrement of the optical absorption, the system was found to be in a terminal state with regard to the rapid phase. On this basis, the formation curve of the intermediate complex was calculated. From the parameters of the curve the maximal saturation of catalase hematins (from horse erythrocytes) by H₂O₂ is 35%. The absolute spectrum of the intermediate complex was established. The variation of the previously calculated rate constant of formation of the intermediate complex was shown to be due to the inapplicability of the pre-steady-state approximation to the rate data. By applying a more general approach and by the use of a computer, the individual rate constants of the peroxidatic scheme were calculated (relevant to micromolar solutions of catalase) k₁ = (3.0 ± 0.2) × 10⁶ M^?1 sec^?1k₄ = (5.6 ± 0.3) × 10⁶ M^?1 sec^?1 These values are 2.2 times higher in a nanomolar solution.     

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.     

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K _m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k _cat) and the k _cat/K _m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s^?1/10,412–10,795 s^?1 M^?1 for AAT-2 zone and 4.6–7.0 s^?1/527–700 s^?1 M^?1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.     

Catalyses by polymer complexes. VII. Enhanced esterolytic reactivity of polymer-coenzyme A,glutathione complexes

The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′_a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, K_CoASH = 1110–2270 M^?1, K_GSH = 170–503M^?1, k′_a,bound at pH 8.65 = 142–341M^?1 sec^?1. k′_a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′_a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant.