Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

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.     

Cobalt(III)-promoted hydrolysis of atp

The rate of phosphate hydrolysis of ATP in the substitution-inert complex Co(NH₃)₄ATP-has been examined in the presence and absence of [Co(cyclen)(H₂O)₂]³⁺. The rate of hydrolysis of Co(NH₃)₄ATP- in the absence of [Co(cyclen)(H₂O)₂]³⁺ is essentially independent of pH in the range 6.0 to 9.0, and the rate constant is 2.6 × 10^?5 sec ^?1 at pH 9.0, 40°C, and 1.0 M ionic strength Rate constants for the hydrolysis of Co(NH₃)₄ATP- in the presence of [Co(cyclen)(H₂O)₂]³⁺ are sharply dependent upon pH in the same range. The rate constants at pH 8.0, 8.6, and 9.0 are 8, 63, and 95 times larger than the rate constant at pH 7.0. At pH 9 the rate constant is 1.2 × 10^?3 sec^?1 for 16 mM Co(NH₃)₄ATP- in the presence of 10 mM [Co(cyclen)(H₂O)₂]³⁺. The proposed mechanism for hydrolysis involves the coordination of a phosphate group of Co(NH₃)₄ATP- by [Co(cyclen)(H₂O)₂]³⁺ to form a dinuclear species, followed by internal attack of coordinated hydroxide on the phosphate chain.     

Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Intramolecular participation of the amide group in the hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate

The rate of hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate (Ib) has been measured in aqueous solution between pH 1 and 6.5 has been found to increase linearly with pH at pH higher than 3. An abnormally large apparent alkaline rate constant of 3.8 × 10⁶M^?1 sec^?1 has been determined. Intramolecular nucleophilic displacement by the amide group at the carbonyl carbon of the ester occurred and a cyclic intermediate was formed. This intermediate has been detected by direct isolation and by measurements of the proton release accompanying the reaction. The rates of hydrolysis of analogous derivatives (IIb-IIIb-IV), for which this intramolecular assistance was not possible, were slower by a factor of about 5 × 10⁵. Such an example of intramolecular catalysis may be useful for a better understanding of the enzymatic catalysis.     

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.     

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.     

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).     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

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.     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

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.     

Kinetics of the acid hydrolysis of heparin from its Cu (II) complex and its relation to biological activity

The acid-catalyzed hydrolysis of heparin from Cu(II) complex was studied as a function of time and temperature. Four independent calculations showed that the hydrolysis, during the 5-hr period examined, obeys the first-order kinetic law. Specific rate constants, calculated at 50°C, 57°C, 65°C, 71°C, and 80°C, were 3.3 × 10^?5 sec^?1, 6.5 × 10^?5 sec^?1, 10.4 × 10^?5 sec^?1, 15.1 × 10^?5 sec^?1, and 26.6 × 10^?5 sec^?1, respectively. Arrhenius plots of the data yielded 14.7 kcal as the energy of activation. An independent run of the self-hydrolysis of heparin at 57°C also obeyed first-order kinetics and its specific rate constant of 6.4 × 10^?5 sec^?1 is in excellent agreement with that of the hydrolysis of Cu(II)-heparin at 57°C. The anticoagulant activity of heparin and of the Cu(II)-heparin are not appreciably different. Further, the inactivation of heparin closely parallels Cu(II) release from the Cu(II) complex which in turn parallels desulfation.     

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

The dehalogenation of the 5-halo-5,6-dihydrouracils: Uracil formation from 5-iodo- and 5-bromo-5,6-dihydrouracil

The elimination of halide ion from either 5-bromo- or 5-iodo-5,6-dihydrouracil to yield uracil is a slow reaction which, in the case of 5-iodo-5,6-dihydrouracil, is 400 times slower than the enzymatic release of ¹²⁵I^? from 5-[¹²⁵I]iodouracil. The elimination of HBr from 5-bromo-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxymethyl)aminomethane (k₂^{Tris base} = 11 × 10^?4M^?1 min^?1, 37°C, ionic strength 1.0 M). At pH values near and above physiological, both the bromo- and iododihydropyrimidines are subject to hydrolysis of the dihydropyrimidine ring, a reaction which parallels halide elimination to yield uracil. The resulting 2-halo-3-ureidopropionate then cyclizes via intramolecular attack of the ureido oxygen atom to yield halide ion and 2-amino-2-oxazoline-5-carboxylic acid as final products. In dilute hydroxide ion, the kinetics of 5-bromo-5,6-dihydrouracil hydrolysis (25°C, ionic strength 1.0 M) show a change in rate-determining step as a function of increasing hydroxide ion concentration, a result which, as in the case of 5,6-dihydrouracil, can be explained in terms of the formation of a tetrahedral addition intermediate. The data are discussed relative to enzymatically catalyzed halopyrimidine dehalogenation.     

Carbonic anhydrase catalyzed oxygen-18 exchange between bicarbonate and water

Oxygen-18 exchange techniques were applied to the dehydration of bicarbonate catalyzed by human carbonic anhydrase C. The rates of depletion of oxygen-18 from labeled bicarbonate were measured for both the catalyzed and uncatalyzed reactions at pH 9.4 and 25 °C. The equilibrium dissociation constant of the enzyme-substrate complex K is 0.321 ± 0.040 m and $\text{k}{}_{\mathrm{<mtext>enz</mtext>}}\text{=}\text{k}{}_2\text{K}{}_{\mathrm{m}}$ is (8.3 ± 1.9) × 10⁵m^?1 sec^?1 under these conditions. On the basis of these results it is demonstrated that the oxygen-18 exchange technique is capable of measuring K and k_enz for the carbonic anhydrase catalyzed dehydration of bicarbonate at a high pH range in which other kinetic techniques are not effective.It was also shown that the oxygen-18 exchange technique is an effective micromethod for the determination of carbonic anhydrase. Rates of isotopic depletion of labeled bicarbonate (in solutions of the enzyme) which fall outside the limits of error for the uncatalyzed rate of depletion demonstrate that this technique can detect concentrations of human carbonic anhydrase C as low as 5 × 10^?11m.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Translational diffusion in the plasma membrane of sea urchin eggs

Translational diffusion in the plasma membrane of individual egg cells from the sea urchin species Paracentrotus lividus has been studied by fluorescence microphotolysis (FM). In order to probe the lipid phase of the membrane, procedures have been worked out by which the fluorescent analog 3,3′-dioctadecyl-oxatricarbocyanine (C₁₈diO) can be incorporated into the membrane. In the unfertilized egg a fraction R = 0.9 of C₁₈diO was mobile having an apparent diffusion coefficient of D = 6.0 × 10^?9 cm² sec^?1. Fifteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 2.7 × 10^?9 cm² sec^?1, respectively. In order to study diffusion of membrane proteins, procedures have been worked out by which the cell surface can be labeled with fluorescein-isothiocyanate (FITC). FITC binds to both the plasma membrane and the vitelline layer. Together with the vitelline layer two-thirds of the FITC-fluorescence could be removed from the egg surface. Gel electropherograms of isolated egg cortices showed various protein bands; however, only two of the protein bands were labeled with FITC. In the unfertilized egg a fraction R = 0.9 of the FITC-labeled membrane proteins was mobile having an apparent diffusion coefficient of D = 35 × 10^?11 cm² sem^?1. Fiteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 7.0 × 10^?11 cm² sec^?1, respectively. FITC-labeled proteins of the fertilization envelope were immobile. Our studies have shown (i) that the egg surface can be fluorescently labeled without blocking fertilization and early development, (ii) that the plasma membrane of unfertilized eggs is a fluid environment permitting a rapid movement of lipids and proteins, and (iii) that after fertilization a substantial degree of lipid and protein mobility is maintained.     

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF₁ at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D_20,w^o = 3.12 × 10^?7 cm² · sec^?1 at 0.05 M, pH 7.8, 20°C, to D_20,w^o = 3.52 × 10^?7 cm² · sec^?1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF₁ behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (R_H = 65 Å).     

Soybeans and radish leaves contain only one of the sulfonium diastereoisomers of S-adenosylmethionine

Using a liquid chromatography method that separates the two sulfonium diastereoisomers of adenosylmethionine, we have found that immature soybeans, soybean callus culture, radish leaves, yeast and rat liver contain only the (S)-sulfonium form of S-adenosylmethionine. Our findings contradict the suggestion by Stolowitz and Minch that 10–20% of naturally-occurring adenosylmethionine may have the (R)-configuration at the sulfonium pole. Absence of the (R)-sulfonium isomer of adenosylmethionine in biological materials indicates that the (R)-sulfonium form of adenosylmethionine present in commercial adenosylmethionine samples is an artifact of the isolation procedure. Our method of measuring the isomers of adenosylmethionine enabled us to readily determine the rate of racemization and hydrolysis of adenosylmethionine. Our rate constants for racemization (K_r) and hydrolysis (K_h) were 2.4 × 10^?6 sec^?1 and 12.3 × 10-^?6 sec^?1, respectively; values which are noticeably different from those of Wu and co-workers which were obtained with a more complicated method (K_r = 8 × 10^?1 sec^?1; K_h = 6 × 10^?6 sec^?1). We believe the absence of the (R)-isomer in vivo is best explained by stabilization of the (S)-isomer as suggested by Wu et al. Although the tissues we have analysed contained the (S)-sulfonium form of adenosylmethionine exclusively, when ethionine-resistant soybean cell lines were given ethionine, they accumulated both sulfonium diastereoisomers of adenosylethionine.