Woodward's Reagent Research Articles

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 m −1 cm −1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.

Potential-dependent interaction of chemical reagents with the gating mechanism of the nerve fiber sodium channel

The action of two water-soluble carbodiimides and of Woodward's reagent K on the properties of the gating mechanism of sodium channels of the Ranvier node membrane was investigated. Treatment with carbodiimide solution (pH 4.8–5.2) at a potential of −80 to −100 mV was shown to delay activation and inactivation of the channels considerably and to reduce the sensitivity of both gating functions to changes in membrane potential. The effective activation charge, determined relative to the limiting logarithmic slope of the activation curve (zef) was reduced by 1.7 times. Treatment of the membrane under the same conditions, but at zero holding potential, induced much smaller changes in properties of the gating mechanism; under these conditions zef remained unchanged. Woodward's reagent at high negative potential induced the same changes in the gating system as carbodiimide at 0 mV. The action of Woodward's reagent also depended on potential, but by a lesser degree than when carbodiimides were used. The results suggest that two types of carboxyl groups exist on the outer surface of the membrane: "mobile," which perform the role of gating particles and which are moved from the surface when the channel changes into the open or inactivated state, and "immobile," which face the external solution whatever the state of the channel.

Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

1. 1. The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. 2. 2. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent K i of 20 μM, suggests that the enzyme presents the “dinucleotide fold” characteristic of most dehydrogenases and kinases. 3. 3. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by α-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of α-oxoglutarate through interaction with one of the carboxyl groups of the substrate. 4. 4. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH 4 +. 5. 5. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.

The presence of essential carboxyl group for binding of cytochrome c in rat hepatic NADPH-cytochrome P-450 reductase by the reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) purified from rat hepatic microsomal fraction was inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a specific agent for modification of carboxyl groups in a protein. The inactivation exhibited pseudo-first order kinetics with a reaction order approximately one and a second-order-rate constant of 0.60 M-1 min-1 in a high ionic strength buffer and 0.08 M-1 min-1 in a low ionic strength buffer. By treatment of NADPH-cytochrome P-450 reductase with EDC, the pI value changed to 6.5 from 5.0 for the native enzyme, and the reductase activity for cytochrome c, proteinic substrate, was strongly inactivated. When an inorganic substrate, K3Fe(CN)6, was used for assay of the enzyme activity, however, no significant inactivation by EDC was observed. The rate of inactivation by EDC was markedly but not completely decreased by NADPH. Also, the inactivation was completely prevented by cytochrome c, but not by K3Fe(CN)6 or NADH. The sulfhydryl-blocked enzyme prepared by treatment with 5,5'-dithio-bis(2-nitrobenzoic acid), which had no activity, completely recovered its activity in the presence of dithiothreitol. When the sulfhydryl-blocked enzyme was modified by EDC, the enzyme in which the carboxyl group alone was modified was isolated, and its activity was 35% of the control after treatment with dithiothreitol. In addition, another carboxyl reagent, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K), decreased cytochrome c reductase activity of NADPH-cytochrome P-450 reductase. These results suggest that the carboxyl group of NADPH-cytochrome P-450 reductase from rat liver is located at or near active-site and plays a role in binding of cytochrome c.

Immobilization of α-amylase on polymeric carriers having different structures

α-Amylase was immobilized onto several polymeric carriers having carboxyl groups by the Woodward's reagent K better than by other methods. The amount of immobilized α-amylase mainly depended on the surface area of carriers, while the enzymic activity depended on the texture of carrier surface. Flat surface was favorable for making the enzyme exhibit high activity and for keeping it active in wide pH range. The durability of immobilized enzyme was excellent at 20°C and pH 5.5.

Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K (WRK). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK. SBP was a strong protector, displaying a dissociation constant ( K d ) of 3 μ m with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 ± 700 m −1 cm −1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.

Relatively simple methodology for the determination of configuration of unsaturation of bacterial monounsaturated fatty acids: application to the unsaturates of Legionella spp.

Unsaturated fatty acids of known degree, position, and configuration of unsturation were esterified, and stereospecifically dihydroxylated at the double bond(s). cis-Hydroxylation was effected using Woodward's reagent (silver acetate/iodine/acetic acid), while trans-hydroxylation was effected using Fenton's reagent (hydrogen peroxide/ferrous sulfate/acetic acid). Diols derived from monounsaturated esters were recovered quantitatively, while tetraols derived from diunsaturates were lost, presumably during extensive washing. The stereospecifically dihydroxylated esters were analyzed by thin-layer chromatography on borate-impregnated silica gel plates, and by gas-liquid chromatography, on a nonpolar capillary column, as the trimethylsilyl, acetyl, n-butylboronyl, isopropylidene, and trifluoroacetyl derivatives. For each derivative, the erythro and threo diols were readily separable, and some resolution of positional isomers was observed. Thus, the cis/trans configuration, and in some instances, the position of unsaturation of the original monounsaturated fatty acid may be deduced. However, gas chromatography-mass spectrometry of appropriately derivatized diol esters is required for unambiguous determination of position of unsaturation in most cases. These reactions are simple, use readily available reagents, and require relatively little operator attention. Further, they do not require specialized apparatus, as do hydrogenation and ozonolysis, or potentially toxic chemicals, such as osmium tetroxide. This series of analyses was applied to the unsaturated non-hydroxylated fatty acids of Legionella species, which were shown to be monounsaturated and of the cis Δ9 family. Position of unsaturation was confirmed by gas chromatography-mass spectrometry.

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding.

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.

Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K 1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K 1 of 5.2 m m and a K 2 of 0.81 min −1. Mg 2+ afforded nearly complete protection with a K d of 2.8 m m. Two moles of [ 14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 m m MgCl 2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the β subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum.

Amino acid residues involved in the action of endo-(1→3)-β- d-glucanase II from Flavobacterium dormitator var. glucanolyticae FA-5

The pH-dependence of the kinetic parameters for the hydrolysis of yeast glucan with endo-(1→3)-β- d-glucanase II from Flav. dormitator var. glucanolyticae FA-5 suggests that two residues (histidyl and carboxyl) are involved in the enzyme action. Chemical modification of the enzyme has been studied in order to identify the kinds and the number of amino acid residues involved in enzyme action. Photo-oxidation and carbethoxylation of the enzyme indicated that the decomposition of a histidine residue is responsible for the loss of activity. Modification of the enzyme with Woodward's reagent K indicated that ∼ 12 carboxyl residues in the enzyme are involved in the catalytic and/or substrate binding-site.

Studies of Bungarus fasciatus venom NAD glycohydrolase. Inhibition, modification, and transglycosidation reactions.

The properties of the NAD-binding domain of Bungarus fasciatus venom NAD glycohydrolase were investigated by a number of biochemical techniques. Inhibitor studies indicated that an intact dinucleotide was required for effective enzyme recognition. Adenosine derivatives, AMP, ADP, ATP, adenosine diphosphoribose (ADP-rib), and 2'-phosphoadenosine diphosphoribose (phospho-ADP-rib), were linear competitive inhibitors of the NADase-catalyzed reaction. Nicotinamide analogs were noncompetitive inhibitors with Ki values that varied with the nature and position of substituent groups. Selective enzyme inactivation by 2,4-pentanedione indicated the importance of a lysinyl residue for NADase activity. The importance of a carboxyl group was also indicated by the sensitivity of the NADase toward N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). Reagents which modified sulfhydryl, histidyl, and arginyl residues were not effective in inactivating B. fasciatus venom NADase. The purified snake venom NADase catalyzed a transglycosidation (pyridine base exchange) reaction. The functioning of a variety of substituted pyridine bases in this reaction was demonstrated by the formation of the corresponding NAD analogs. Kinetic studies of the transglycosidation reaction were consistent with the partitioning of an enzyme-ADP-rib intermediate between water and the substituted pyridine base. Concentrations of pyridine bases required for enzyme inhibition differed significantly from those needed for the pyridine base exchange reaction, suggesting two sites or two forms of the NADase differing in affinity for pyridine bases. A number of NAD analogs were also shown to be effective ribosyl donors in the transglycosidation reaction.

An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase.

Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides. Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide binding site of the reductase.

Reaction of Woodward's reagent K with pancreatic porcine phospholipase A 2: Modification of an essential carboxylate residue

Summary Reaction between N-ethyl-5-isoxazolium-3-sulfonate, a reagent specific for carboxylate residues, and pancreatic porcine phospholipase A 2 results in complete elimination of enzymatic activity. The modification reaction is prevented in presence of hexadecylphosphorylcholine and N-palmitoylaminoethylphosphorylcholine acting as reversible phospholipase A 2 inhibitors. The decrease of enzymatic activity correlates with the modification of a single carboxylate residue. The possible catalytic role of the carboxylate is discussed and a model for the active-site is proposed.

Action pattern and active site of endo .BETA.-1,3-glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5.

The action pattern and amino acid residues involved in the active site of endo β-1, 3-glucanase IV from Flav. dormitator var. glucanolyticae FA-5 were investigated. The glucanase was specific for straight chains of β-1, 3-glucosidic linkages. The enzyme endowisely hydrolyzed β-1, 3-ghicans to produce laminaripentaose and showed transglucanosylation activity. The glucanase was inactivated by modification of histidine residues with diethylpyrocarbonate and by photooxidation, and by modification of carboxyl residues with Woodward's reagent K and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The presence of β-1, 3-ghicans resulted in protection of the enzyme from inactivation by modifications, suggesting that these residues are at the β-1, 3- glucan binding and/or active site of the glucanase molecule.

Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3

Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme. The inactivation rates followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site. ADP and ATP strongly protected the thermophilic ATPase against both reagents. GDP and IDP protected less, whilst CTP did not protect. Experiments in which the incorporation of [ 14C]phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8–9 mol [ 14C]phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.

Synthese De La N‐acetylmuramyl (OXO‐14C‐propyl)‐L‐alanyl‐D‐isoglutamine (MDP‐14C)

AbstractSYNTHESIS OF N‐ACETYLMURAMYL (14C‐OXOPROPYL)‐L‐ALANYL‐D‐ ISOGLUTAMINE (14C ‐ MDP)Condensation of (1‐14C) DL‐2‐bromopropionic acid with benzyl 2‐acetamido‐4,6‐0‐benzylidene‐2‐deoxy‐β‐D‐glucopyranoside 7 in presence of sodium hydride gave 50 % of crystalline compound 3, having the correct stereochemistry of natural muramic acid. The latter, in presence of Woodward's reagent, reacted with L‐alanyl‐D‐isoglutamine benzyl ester hydrochloride, leading to compound 4. Removal of benzylidene and benzyl groups by successive acetic acid exposure and hydrogenolysis gave rise to 2‐acetamido‐2‐deoxy‐3‐0 (2‐D‐(14CO) propionyl‐L‐alanyl‐D‐isoglutamine)‐D‐glucopyranose (14C‐MDP) Specific activity : 42 mCi/mMole in a 10 % overall yield based on sodium (1‐14C) propionate.

An essential carboxylic acid group in human prostate acid phosphatase

Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K ( N- ethyl-5-phenylisoxazolium-3′-sulfonate ) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and l-(+)-tartrate, but not in the presence of non-inhibitory D-(−)-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of d- and of l-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.

A homogeneous isoenzyme of human liver acid phosphatase

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K i is 4.3 × 10 −7 m at pH 4.8 in 50 m m sodium acetate/100 m m sodium chloride. K i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K i for l-(+)-tartrate. The pH dependence of both K m and V indicate the effect of substrate ionization ( pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.

Reaction of α-mannosidase from Phaseolus vulgaris with group-specific reagents. Essential carboxyl groups

When the pK m of α-mannosidase was determined at different pH values, the results indicated that ionizable groups with pK values of approx. 3.8 and 5.7 could be essential. Modification with carbodiimide or Woodward's Reagent K abolished the enzyme activity. The substrate analogue, α-methyl- d-mannoside, protected the enzyme against inactivation. Incorporation of a 14C-labeled nucleophile reagent in the presence or absence of the analogue suggested that 2–4 carboxyl groups were protected. Exchange studies indicated that the essential Zn 2+ could be bound to such groups. There was no indication that hydroxyl groups, sulphydryl groups, guanidino groups or amino groups take part in the catalytic activity.

Calf-Spleen Nicotinamide-Adenine Dinucleotide Glycohydrolase. Properties of the Active Site

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.