Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Ferulic acid: a substrate for two isoperoxidases from Nicotiana tabacum tissue cultures

An anodic isoperoxidase (A₂) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C₄) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H₂O₂. When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A₂ and C₄ appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The Kms for guaiacol are 4 and 4·5 mM for isoperoxidases C₄ and A₂, respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.     

Double-helical structures for polyxanthylic acid

Polyxanthylic acid has been found to exist in two different duplex forms, A_I and A_II. a_I, formed at pH5·7, occurs in a compact lattice with nearest neighbor molecules spaced at 2.11 nm. It has an axial translation per residue, h = 0·301 nm, and a rotation per residue, t = 36·0 °. The intensity distribution in its X-ray fiber diffraction pattern is analogous to that of A-RNA (h = 0·281 nm, t = 32·7 °). On the other hand A_II, formed at pH 8·0, has a less compact, statistically disordered crystal packing with nearest neighbors 2·35 nm apart. It has h = 0·252 nm and t = 32·7 ° and gives an X-ray intensity distribution essentially identical to A-DNA (h = 0·256 nm, t = 32·7 °). Similar right-handed helical duplex models, with flexible C-3′-endo sugar rings, have been developed for each molecular structure. Both have purine purine base-pairs, possibly triply hydrogen-bonded, and certainly with the same symmetry as Watson-Crick pairs but with a 0·2 nm greater C-1′ … C-1′ separation.     

The kinetics of intrinsic factor-vitamin B12 binding

The kinetic behaviour of intrinsic factor-vitamin B₁₂ binding has been examined under varying conditions using an albuminised charcoal separation technique. The overall reaction obeys second order rate laws. The intrinsic factor considered alone obeys first order laws; the velocity of reaction of vitamin B₁₂ is too fast for measurement by the technique described but by deduction obeys first order laws. Rate constants as three temperatures, (k₂ at 25°C=1.56·10⁸·mole^?1·s^?1) the activation energy (E=12.7 kJ·mole^?1) and Arrhenius constant (A=2.7·10¹⁰ 1·mole^?1·s^?1 have been calculated. There is the possibility of diffusion control of the reaction in which case the E and A values are invalid. The effect of pH on the reaction has been studied and the results discussed in relation to the pH studies of other workers whose results show disagreement. Albumin coated charcoal was shown to discriminate between intrinsic factor-vitamin B₁₂ and free vitamn B₁₂ over a wide pH range. The apparent under-estimation of intrinsic factor in dilute solution was shown to be due to adsorption of the intrinsic factor to plastic tubes.     

The transformation of chlorophenols by lactoperoxidase

The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H₂O₂ ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν₀) was only slightly influenced by changes in [H₂O₂] ? 5 μM. ν₀ responded to [chlorophenol] according to the empirical expression ν₀ = [lactoperoxidase]·(k₁[chlorphenol] + k₂[chlorophenol]²). The constant k₁ was found to be 5.8 · 10⁵, 1.8 · 10⁶, 1.9 · 10⁶ M^?1 · s^?1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H₂O₂ and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products.     

Crystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61.

A pencillin-sensitive enzyme, the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (M_r = 6000 to 7500) solution at pH 7·6. X-ray examination of the orthorhombic crystals shows the space group is P2₁2₁2₁, with unit cell dimensions $\text{a = 51·1}\text{A}\text{?}\text{, b = 67·4}\text{A}\text{?}\text{,}\text{and}\text{c = 102·9}\text{A}\text{?}$. With four molecules of molecular weight 38,000, the $\text{A}\text{?}{}^3\text{/}\text{dalton}$ ratio for the cell is 2·33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2·4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å.     

An assay for botulinum toxin types A, B and F that requires both functional binding and catalytic activities within the neurotoxin

Aim: To develop a novel assay technique for the botulinum neurotoxin family (BoNTs) which is dependent on both the endopeptidase and receptor‐binding activities of the BoNTs and which is insensitive to antigenic variation with the toxin family. Methods and Results: An endopeptidase activity, receptor‐binding assay (EARB assay) has been developed which captures biologically active toxin from media using brain synaptosomes. After capture, the bound toxin can be incubated with its substrate, and cleavage detected using serotype‐specific antibodies raised against the cleaved product of each toxin serotype. The EARB assay was assessed using a range of BoNT serotypes and subtypes. For BoNT/A, detection limits for subtypes A₁, A₂ and A₃ were 0·5, 3 and 10 MLD₅₀ ml^?1, respectively. The limit of detection for BoNT/B₁ was 5 MLD₅₀ ml^?1 and a novel antibody‐based endopeptidase assay for BoNT/F detected toxin at 0·5 MLD₅₀ ml^?1. All these BoNTs can be captured from media containing up to 10% serum without loss of sensitivity. BoNT/A₁ could also be detected in dilutions of a lactose‐ containing formulation similar to that used for clinical preparations of the toxin. Different serotypes were found to possess different optimal cleavage pHs (pH 6·5 for A₁, pH 7·4 for B₁). Conclusions: The EARB assay has been shown to be able to detect a broad range of BoNT serotypes and subtypes from various media. Significance and Impact of the Study: The EARB assay system described is the first convenient in vitro assay system described which is requires multiple functional biological activities with the BoNTs. The assay will have applications in instances where it is essential or desirable to distinguish biologically active from inactive neurotoxin.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

6-Halogenopyridin-2-olato complexes with the diruthenium(2+) core: Equilibria between head-to-head and head-to-tail structures

The dinuclear bis(6-X-pyridin-2-olato) ruthenium complexes [Ru₂(μ-XpyO)₂(CO)₄(PPh₃)₂] (X = Cl (4B) and Br (5B)), [Ru₂(μ-XpyO)₂(CO)₄(CH₃CN)₂] (X = Cl (6B), Br (7B) and F (8B)) and [Ru₂(μ-ClpyO)₂(CO)₄(PhCN)₂] (9B) were prepared from the corresponding tetranuclear coordination dimers [Ru₂(μ-XpyO)₂(CO)₄]₂ (1: X = Cl; 2: X = Br) and [Ru₂(μ-FpyO)₂(CO)₆]₂ (3) by treatment with an excess of triphenylphosphane, acetonitrile and benzonitrile, respectively. In the solid state, complexes 4B-9B all have a head-to-tail arrangement of the two pyridonate ligands, as evidenced by X-ray crystal structure analyses of 4B, 6B and 9B, in contrast to the head-to-head arrangement in the precursors 1-3. A temperature- and solvent-dependent equilibrium between the yellow head-to-tail complexes and the red head-to-head complexes 4A-7A and 9A, bearing an axial ligand only at the O,O-substituted ruthenium atom, exists in solution and was studied by NMR spectroscopy. Full ¹H and ¹³C NMR assignments were made in each case. Treatment of 1 and 2 with the N-heterocyclic carbene (NHC) 1-butyl-3-methylimidazolin-2-ylidene provided the complexes [Ru₂(μ-XpyO)₂(CO)₄(NHC)], X = Cl (11A) or Br (12A). An XRD analysis revealed the head-to-head arrangement of the pyridonate ligands and axial coordination of the carbene ligand at the O,O-substituted ruthenium atom. The conversion of 11A and 12A into the corresponding head-to-tail complexes was not possible.     

Mechanism of primary and secondary ion-radical pair formation in photosystem I complexes

The mechanisms of the ultrafast charge separation in reaction centers of photosystem I (PS I) complexes are discussed. A kinetic model of the primary reactions in PS I complexes is presented. The model takes into account previously calculated values of redox potentials of cofactors, reorganization energies of the primary P700⁺A ₀ ^- and secondary P700⁺A ₁ ^- ion-radical pairs formation, and the possibility of electron transfer via both symmetric branches A and B of redox-cofactors. The model assumes that the primary electron acceptor A₀ in PS I is represented by a dimer of chlorophyll molecules Chl2A/Chl3A and Chl2B/Chl3B in branches A and B of the cofactors. The characteristic times of formation of P700⁺A ₀ ^- and P700⁺A ₁ ^- calculated on the basis of the model are close to the experimental values obtained by pump-probe femtosecond absorption spectroscopy. It is demonstrated that a small difference in the values of redox potentials between the primary electron acceptors A_0A and A_0B in branches A and B leads to asymmetry of the electron transfer in a ratio of 70: 30 in favor of branch A. The secondary charge separation is thermodynamically irreversible in the submicrosecond range and is accompanied by additional increase in asymmetry between the branches of cofactors of PS I.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

Subunit Structure of Soybean 11S Globulin

The acidic and the basic subunits were shown to be present in equimolar amounts in the 11S globulin molecule by the densitometric scanning of the SDS gel and the molecular weight consideration. The four acidic subunits (A₁, A₂, A₃ and A₄) were found to be present in the approximate molar ratio of 1:1:2:2. Four basic subunits separated and designated as B₁, B₂, B₃ and B₄ based on the relative mobilities in the acidic gel in 7 m urea were found to be present in the approximate molar ratio of 1:1:2:2. The four basic subunits were fractionated in approximately same amounts into three different peaks, peak I (B₁ and B₂), peak II (B₃) and peak III (B₄) by CM-Sephadex C–50 column chromatography in the presence of 6 m urea. Three kinds of intermediary subunits of 11S globulin were fractionated with DEAE-Sephadex A–50 in the absence of reducing agents in 6 m urea, and disulfide bonds appeared to participate in the binding between the acidic and the basic subunits in the molar ratio of 1: 1 with the following combinations; A₁ and A₂ combined with B₃, A₃ with B₁ and B₂, and A₄ with B₄. In view of the above results and molecular weight consideration, a new model of subunit structure was proposed for 11S globulin.     

Preliminary crystallographic study of macromomycin: The apoprotein of the antitumor antibiotic auromomycin

Macromomycin (M_r 12,000) is the apoprotein of the antitumor drug auromomycin, which inhibits DNA synthesis by causing single-strand breaks in DNA. Two orthorhombic crystal forms of macromomycin have been observed. The platelike crystals of one form belong to space group P2₁2₁2₁, with cell dimensions $\text{a = 48·92}\text{A}\text{?}\text{, b = 54·71}\text{A}\text{?}\text{, c = 103·31}\text{A}\text{?}\text{, Z = 8}$. The crystals of the second form are needle-shaped, and belong to space group P2₁2₁2, with cell dimensions $\text{a = 46·1}\text{A}\text{?}\text{, b = 54·4}\text{A}\text{?}\text{, c = 41·2}\text{A}\text{?}\text{, Z = 4}$. At this point in time, the platelike crystals appear the most suitable for continued crystallographic studies.     

FOUR PROTEIN POLYMORPHISMS IN PIGEON BRAIN EXTRACTS DETECTED BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Abstract— Proteins of the brain extracts of 85 individual pigeons (Columba livia) were mapped by two-dimensional gel electrophoresis. The method is a modification of O'Farrell 'S technique and separates proteins first by charge and then by molecular weight. There were three proteins, A, B and D which had each a variant form. The positions of these six proteins on the gel corresponded to the following pH values and molecular weight values: protein A₁, 6.4/43,000; A₂, 6.6/43,000; B₁, 5.7/41,000; B₂, 5.8/40,000; D₁, 6.2/22,000; D₂, 6.2/21,000. The variants are genetically determined, since protein A, B and D each occurred in three phenotypes (A₁, A₁A₂ and A₂; B₁, B₁B₂ and B₂; D₁, D₁D₂ and D₂) corresponding to the three possible genotypes. From the observed frequencies of the phenotypes the following allele frequencies were calculated: allele A₁, 72%; A₂, 28%; B₁, 15%; B₂, 85%; D₁, 74%; D₂, 26%. A fourth protein named C occurred in four different forms (C₁, 7.2/37,000; C₂, 7.2/36,000; C₃, 7.1/37,000; C₄, 7.1/36,000) and six phenotypes (C₁, C₁C₂, C₂, C₁C₃, C₂C₃ and C₄C₃). This polymorphism is also interpreted as being genetically determined. The four alleles coding for the four protein C forms had the following frequencies: allele C₁, 62%; C₂, 27%; C₃, 10.5%; C₄, 0.5%.     

Efficacy of pyramiding elite alleles for dynamic development of plant height in common wheat

Plant height is an important botanical feature closely related to yield. Two populations consisting of 118 and 262 accessions respectively were used to identify elite alleles for plant height and to validate their allelic effects. Plant height was measured from the early booting to the flowering stages. Simple sequence repeat markers for candidate quantitative trait locus (QTL) regions with large effects identified in a doubled haploid (DH) population (Hanxuan 10 × Lumai 14) were selected for further verification by association analysis. Nine loci significantly (P < 0.001) associated with plant height were detected 13 times in the population with 118 accessions. Three loci (Xgwm11-1B, Xwmc349-4B and Xcfd23-4D) were identified in three, two and two periods of plant height growth, respectively. Markers Xbarc168-2D, Xgwm249-2D, Xwmc349-4B, Xcfd23-4D and Xgwm410-5A located at or near additive QTL regions in the DH population proved to coincide with known Rht loci. The results showed a consistency between linkage analysis and association mapping, and also confirmed the value of fine mapping of QTL through combined linkage and association analyses. For final plant height, the alleles Xgwm11-1B ₂₀₈ , Xwmc349-4B ₁₀₃ and Xcfd23-4D ₂₀₂ exhibited negative effects, i.e. reducing plant height; Xwmc349-4B ₁₀₁ and Xcfd23-4D ₂₀₅ showed significant positive effects. A second larger population (262 accessions) was used to validate the effects of these large-effect alleles and the efficacy of pyramiding in eight environments (year × site × water regime combinations). Strong correlations between final plant height and numbers of large-effect alleles indicated that the alleles contributed additively to plant height. The additive effects showed that pyramiding elite alleles for target traits has significant potential for wheat breeding.     

Structure and magnetic properties of a tetranuclear Cu4O4 open-cubane in [Cu(L)]4·4H2O with L = (E)-N′-(2-oxy-3-methoxybenzylidene)benzohydrazide

The in-situ formed hydrazone Schiff base ligand (E)-N′-(2-oxy-3-methoxybenzylidene)benzohydrazide (L²⁻) reacts with copper(II) acetate to a tetranuclear open cubane [Cu(L)]₄ complex which crystallizes as two symmetry-independent (Z′ = 2) S₄-symmetrical molecules in different twofold special positions with a homodromic water tetramer. The two independent (A and B) open- or pseudo-cubanes with Cu₄O₄ cores of 4 + 2 class (Ruiz classification) each have three different magnetic exchange pathways leading to an overall antiferromagnetic coupling with J_1B = J_2B = −17.2 cm⁻¹, J_1A = −36.7 cm⁻¹, J_2A = −159 cm⁻¹, J_3A = J_3B = 33.5 cm⁻¹, g = 2.40 and ρ = 0.0687. The magnetic properties have been analysed using the H = −Σ_i,jJ_ij(S_iS_j) spin Hamiltonian.     

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A_2B receptor antagonists because of its slight selectivity against A₁ and A_2A receptors and modest A_2B potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A_2B potency, modest selectivity versus A_2A and A₁ receptors, and good rodent PK properties.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.