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PubMed1996

Chemical modification and inactivation of rat liver arginase by N-bromosuccinimide: reaction with His141.

Treatment of rat liver arginase with N-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzynska (1979) Acta Biochim. Pol. 26, 103-114). In order to probe the chemistry of N-bromosuccinimide inactivation and the role of tryptophan residues in catalysis, the two tryptophan residues of rat liver arginase, Trp122 and Trp164, have been separately mutated to phenylalanine using site-directed mutagenesis of the protein expressed in Escherichia coli. Both single Trp -> Phe mutant enzymes have kinetic parameters nearly identical to those for the wild-type enzyme. Treatment of native, wild-type, and each of the Trp -> Phe mutant enzymes with N-bromosuccinimide results in loss of absorbance at 280 nm and is accompanied by a loss of catalytic activity. However, treatment of the wild-type enzyme with N-bromosuccinimide in the presence of the arginase inhibitors NG-hydroxy-L-arginine or the combination of L-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases with N-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated by N-bromosuccinimide, indicating that His141 is the critical target for the N-bromosuccinimide inactivation of the enzyme.

Amino Acid Sequence↗

PubMed1975

Bacillus cereus beta-lactamase. Reaction with N-bromosuccinimide and the properties of the product.

The effect of N-bromosuccinimide on the enzymatic activity and the conformation of a Bacillus cereus beta-lactamase (penicillin amido-beta-lactamase EC 3.5.2.6) was studied. Incubation with 10 muM N-bromosuccinimide caused over 95% decrease of the enzymatic activity within 15 min. Spectrophotometric titration with N-bromosuccinimide showed that the reaction proceeded in two steps. The half-inactivated enzyme was prepared by the reaction with N-bromosuccinimide and its properties examined. Amino acid analysis showed that the half-inactivated enzyme contained one residue of tryptophan less while other amino acid contents were similar. Neither the molecular weight nor the mobility in disc electrophoresis was changed. However, the affinity to a cephalexin-CH-Sepharose column was increased, and the Km value for cloxacillin was one-third that of the native enzyme, although that for benzylpenicillin was similar. These results indicate that a tryptophan residue sensitive to N-bromosuccinimide is essential for the maintenance of the rigid conformation and that its oxidation alters the enzyme in a manner such that a substrate with a bulky group in its side chain can form an enzyme-substrate complex more easily. In the native enzyme, the value of (f(a))(eff) (Lehrer, S.S. (1971) Biochemistry 10, 3254-3263), did not vary significantly in the absence or the presence of cloxacillin. In contrast, in the half-inactivated enzyme the presence of cloxacillin affected the conformation such that over two thirds of the tryptophyl fluorescence were accessible for quenching by KI, although about half was accessible in the absence of cloxacillin.

Bacillus cereus↗

PubMed1977

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide.

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate alpha-N-benzoyl-L-arginine ethl ester were studied in the region of pH 3.5-9.0. All substrates (and inhibitors) test, with the exception of alpha-N-benzyoyl-L-arginine p-nitroanilide, displayed approximately a two fold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is probably Trp-177.

Amino Acids↗

PubMed1998

Kinetics of inactivation of Penaeus penicillatus acid phosphatase during inhibition by N-bromosuccinimide.

In the present investigation, the inactivation by N-bromosuccinimide of acid phosphatase from penaeus penicillatus has been studied using the kinetic method of the substrate reaction during modification of enzyme activity as previously described by Tsou [(1988, Adv. Enzymemol. Related Areas Mol. Biol. 61, 381-436]. The results show that inactivation of the enzyme by N-bromosuccinimide is a slow, reversible reaction. The results also clearly show that the modification of the tryptophan residues of penaeus penicillatus acid phosphatase by high concentrations of N-bromosuccinimide led to the complete inactivation of the enzyme. The microscopic rate constants were determined for the reaction of the inactivator with the free enzyme and with the enzyme-substrate complex. Comparison of the obtained microscopic rate constants indicates that the presence of the substrate offers marked protection of the enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and may be situated at the active site of the enzyme.

Acid Phosphatase↗

PubMed1977

Studies of the N-bromosuccinimide inactivation of the enzyme rhodanese.

The enzyme rhodanese (Thiosulfate: cyanide sulphurtransferase, EC 2.8.1.1) is rapidly inactivated by treatment with N-bromosuccinimide. Spectrophotometric titration and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that neither tryptophan oxidation nor polypeptide chain cleavage can account for the inactivation. Sulfhydryl group assays using the colormetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) after destruction of excess N-bromosuccinmide, indicate that approximately 2 sulfhydryl groups per enzyme molecule are lost. Further, rhodanese inactivated by N-bromosuccinimide can be reactivated (approximately 95%) by incubation with the substrate thiosulfate. It is postulated that N-bromosuccinimide inactivates rhodanese by inducing the formation of a disulfide bond involving the active site sulfhydryl group of the enzyme and a second sulfhydryl group which can be brought close to the active site in the flexible native structure.

Binding Sites↗

PubMed1976

Formation of delta1-acetoxytryptophan-62 in the oxidation of tryptophan-62 of hen egg-white lysozyme by N-bromosuccinimide in acetate buffer.

The reaction of equimolar amounts of N-bromosuccinimide and hen egg-white lysozyme in acetate buffer, under the conditions of Hayashi et al. (Hayashi, K., Imoto, T., Funatsu, G., and Funatsu, M. (1965), J. Biochem. (Tokyo) 58, 227), yields a protein mixture that has a time-dependent 13C-NMR spectrum. The initial natural-abundance 13C-NMR spectrum indicates the presence of about equal amounts of [oxindolealanine-62]lysozyme and [delta1-acetoxytryptophan-62]lysozyme. The latter converts to [oxindolealanine-62]lysozyme with a half-life of about 2 days at 25 degrees C and pH 3.9. Two observations indicate that the source of the acetyl group of delta1-acetoxytryptophan-62 is the acetate buffer. First, the spectrum of a lysozyme sample treated with N-bromosuccinimide in the presence of [1-13C]acetate yields a very strong acetyl ester carbonyl resonance. The time dependence of the intensity of this resonance yields a half-life of 44 h for [delta1-acetoxytryptophan-62]lysozyme. Second, the initial natural-abundance 13C-NMR spectrum of a lysozyme sample treated with N-bromosuccinimide in the absence of acetate indicates essentially complete conversion of tryptophan-62 into oxindolealanine.

Acetates↗

PubMedDate not supplied

Reactivity of 3-HBA-6-hydroxylase with diethylpyrocarbonate and N-bromosuccinimide: effect of chemical modifications on kinetic and spectral properties of the enzyme.

The rapid inactivation of 3-HBA-6-hydroxylase by 100 microM diethylpyrocarbonate or 40 microM N-bromosuccinimide and protection offered by the substrate, 3-hydroxybenzoate, against these chemical modifications implicate the involvement of histidine and tryptophan in the catalytic activity of the enzyme. Inactivation of the enzyme by diethylpyrocarbonate followed pseudo-first-order kinetics, and an "n" value of 1.3 was obtained. Inactivation of the enzyme by N-bromosuccinimide was instantaneous and failed to follow pseudo-first-order kinetics. Distinct and incremental changes in the UV absorption, emission fluorescence, and near UV-CD spectra of the enzyme upon its titration with increasing concentrations of diethylpyrocarbonate or N-bromosuccinimide may be ascribed to modification and/or changes in the microenvironment of aromatic amino acid residue(s) such as tryptophan in the enzyme.

Bromosuccinimide↗

PubMed2006

Spectrophotometric determination of olanzapine by its oxidation with N-bromosuccinimide and cerium(IV)sulfate.

Three simple spectrophotometric methods have been described for the assay of olanzapine in its pure and pharmaceutical formulations. The direct method (A) is based on the drug oxidation with excess of N-bromosuccinimide in acidic medium and the two indirect methods (B and C) are based on the oxidation of the drug with excess of N-bromosuccinimide and cerium(IV)sulfate, followed by the reaction of the unconsumed oxidants with celestine blue. The calibration graphs were linear over the range 10 - 120 microg mL(-1) (method A), 0.5 - 6.0 microg mL(-1) (method B) and 0.6 - 3.0 microg mL(-1) (method C). After validation, the proposed methods were successfully applied to assay of olanzapine in its commercial tablets with mean percentage recoveries of 101.23 +/- 0.10, 96 +/- 0.10 and 94 +/- 0.04%. The mechanism of olanzapine oxidation with N-bromosuccinimide was also proposed.

Benzodiazepines↗

PubMed2004

Synthesis of functionalized 1-azaspirocyclic cyclopentanones using bronsted acid or N-bromosuccinimide promoted ring expansions.

Azaspirocyclic ring systems are present in a variety of alkaloids. Functionalized 1-azaspirocyclopentanones (6, 7, 11, 12) can be efficiently constructed through semipinacol ring expansion reactions of 2-(1-hydroxycyclobutyl)-p-toluenesulfonylenamides (4) promoted by either a Bronsted acid ((S)-(+)-10-camphorsulfonic acid or HCl) or N-bromosuccinimide, an electrophilic bromine source. Reactions promoted by N-bromosuccinimide tend to proceed in higher yields (80-95%) and with greater diastereoselectivity (3:1-1:0) compared to those reactions promoted by a Bronsted acid. In addition, N-bromosuccinimide promoted reactions can produce a complementary stereochemical outcome compared to the reactions using Bronsted acid.

Journal Article↗

PubMed1996

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide.

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.

Alkaline Phosphatase↗

PubMed1986

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide.

Reaction of N-bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N-bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.

Animals↗

PubMed1981

Selective N-bromosuccinimide oxidation of the nonfluorescent tryptophan-31 in the active center of thioredoxin from Escherichia coli.

The two tryptophan residues (Trp-28 and Trp-31) of thioredoxin-S2 from Escherichia coli were selectively tritiated with trifluoroacetic [3H]acid. The 3H label was introduced to permit quantitative amino acid sequence analyses of the result of N-bromosuccinimide oxidation of tryptophan to oxindolylalanine. Addition of 3-fold molar excess of N-bromosuccinimide at pH 4 modifies a tryptophan in thioredoxin-S2 that is nonessential for enzyme activity with thioredoxin reductase and has a strongly quenched fluorescence in both oxidized and reduced thioredoxin. This residue was shown to be Trp-31 by amino acid sequence analyses of 3H-labeled chymotryptic peptides from the modified protein. The results demonstrate that the second tryptophan residue, Trp-28, signals a conformational change on reduction of the active-center disulfide to a dithiol by increasing its fluorescence quantum yield about 6-fold at pH 7. The differential reactivity of the tryptophan residues agrees with the known three-dimensional structure of thioredoxin-S2.

Amino Acid Sequence↗

PubMed1983

[Interaction of DNA with N-bromosuccinimide. G+C-specific reaction potentially useful for structural and functional studies].

Incomplete modification of DNA fragments labeled at one terminus with N-bromosuccinimide is shown to be useful for localization of G and C residues along the polynucleotide chain. After N-bromosuccinimide treatment the fragments can be split at modified residues with piperidine. The rate of the reaction of the single-stranded fragments is extremely fast and much higher than that of double-stranded DNA. The modification can be used for structure and function studies on DNA.

Base Sequence↗

PubMed1975

Modification of bovine alpha-lactalbumin with N-bromosuccinimide and 2-hydroxy-5-nitrobenzylbromide.

Reaction of alpha-lactalbumin at pH 7 in aqueous solution with either 2-hydroxy-5-nitrobenzylbromide or N-bromosuccinimide yields derivatives in which only 2 of the 4 tryptophan residues are modified. All 4 residues of tryptophan are modified under the similar conditions in 8 M urea. Structural analysis of the modified derivatives revealed that tryptophans 26 and 118 are the sole reactive residues and that tryptophan 118 reacts more rapidly than tryptophan 26. The fluorescence of alpha-lactalbumin modified to varying extents with N-bromosuccinimide indicates that tryptophan 118 is exposed to solvent whereas tryptophan 26 is in a more hydrophobic environment. The chemical reactivities and fluorescence properties of tryptophans 26 and 118 are consistent with the proposed conformations of alpha-lactalbumin based on its similarity with egg white lysozyme. The kinetic properties of both derivatives of alpha-lactalbumin containing up to 2 modified residues indicate that each derivative has decreased affinity for the galactosyltransferase but that at saturating concentrations, Km and Vmax for lactose synthesis are unchanged from those of native alpha-lactalbumin.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed1975

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.

Amino Acids↗

PubMed1976

N-bromosuccinimide assay of penicillins and cephalosporins.

All penicillins and cephalosporins known to possess biological activity respond to an N-bromosuccinimide assay. The developed method is not yet usable for determining stability, but it is useful as a bulk or batching assay.

Bromosuccinimide↗

PubMed1996

Discrimination between the four tryptophan residues of MM-creatine kinase on the basis of the effect of N-bromosuccinimide on activity and spectral properties.

Rabbit muscle cytosolic creatine kinase (MM-CK) has been treated with N-bromosuccinimide, a reagent known to oxidize selectively the indole moiety of tryptophan residues of proteins in acidic conditions. Inactivation of the enzyme is achieved by modification of one residue per monomer. NBS treatment decreases the ultraviolet absorbance at 280 nm and the intrinsic fluorescence of the protein. From these data it can be deduced that the quantum yields of the four tryptophan residues of each monomer are different due to the more or less hydrophobic environment of each of them and that at least two of them are sufficiently close to Cys 282 to allow fluorescence energy transfer to an extrinsic fluorophore bound to this residue. The accessibility to iodide of the tryptophans has been evaluated during guanidinium chloride denaturation. These data allowed us to acquire a new insight into the environment, the contribution to intrinsic fluorescence and the role in enzymatic activity and fluorescence resonance energy transfer of the tryptophan residues of CK and to tentatively assign a position in the sequence to each of them.

Animals↗

PubMed1980

Modification of 50S ribosomal subunits with N-bromosuccinimide.

The 50S subunits of Escherichia coli ribosomes were modified with the tryptophan reagent N-bromosuccinimide, and the sulfhydryl groups, the modification of which is accompanied by stimulation of polypeptide synthesis (López-Rivas, A. et al. (1978) Eur. J. Biochem. 92, 121), were regenerated by incubation with simple thiols. This treatment inactivates poly(U)-dependent polyphenylalanine synthesis, peptidyl transferase and elongation factor G-dependent GTPase. Incubation with proteins from untreated 70S ribosomes produces partial reactivation of polyphenylalanine synthesis and GTPase activity. Modification is accompanied by loss of 4-5 tryptophan residues per subunit.

Bromosuccinimide↗