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At least 19 recordsLinked to original sources
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↗
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↗
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↗
PubMed1976

Studies on the subsite structure of amylases. IV. Tryptophan residues of glucoamylase from Rhizopus niveus studied by chemical modification with N-bromosuccinimide.

Chemical modification of glucoamylase [EC 3.2.1.3] from Rhizopus niveus by N-bromosuccinimide was carried out to investigate the role of tryptophan residues in the enzyme-catalyzed reaction and their location in the enzyme subsites. Of the ten tryptophan residues of the enzyme four could be modified. The two more reactive residues were confirmed not be essential for the catalytic activity for the hydrolysis of maltodextrin and phenyl alpha-maltoside. Complete loss of the catalytic activity, however, was brought about by modifying the two less reactive residues, and the modification of these residues was prevented by the substrates. The characteristic difference spectrum produced by maltose (7) disappeared in parallel with the loss of the catalytic activity. These results suggest that the tryptophan residue(s) responsible for the maltose-induced difference spectrum may be located at one of the subsite near the catalytic site and plays an important role in the catalytic activity of the enzyme.

Binding Sites↗
PubMed1977

Kinetic studies on the chemical modification of lysozyme by N-bromosuccinimide and its protection by substrates and analogs.

The chemical modification of tryptophan residues of hen egg-white lysozyme by N-bromosuccinimide (NBS) was studied kinetically by the stopped-flow method, monitoring changes in absorbance and fluorescence. One most rapidly reacting tryptophan residue, probably Trp 62, was clearly distinguished from four other residues in terms of rate of modification. This residue was protected by ethylene glycol chitin, N-acetyl glucosamine (NAG), and tri-NAG, but not by gluconolactone. The dissociation constant Kd of the enzyme-ligand complex was obtained from the protection effects. These results are in good agreement with results previously obtained.

Acetylglucosamine↗
PubMed1978

Tryptophan residues of saccharifying alpha-amylase from Bacillus subtilis. A kinetic discrimination of states of tryptophan residues using N-bromosuccinimide.

Four tryptophan residues of saccharifying alpha-amylase from B. subtilis out of eleven in total are reactive towards N-bromosuccinimide (NBS), suggesting that they are on the surface of the enzyme. This is consistent with the results of solvent perturbation difference spectrophotometry with ethylene glycol. One of four tryptophan residues was clearly distinguished from the other three in reactivity with NBS by the stopped-flow method. This most reactive tryptophan residue was not protected from modification by substrates of analogs, indicating that the tryptophan is not located in the substrate binding site. One of the other three tryptophan residues, probably the second most reactive one, is considered to be related in some way to the glycosyl transfer in the reaction of the enzyme with maltose as a substrate.

Amylases↗
PubMed1976

Cleavage of human serum transferrin with N-bromosuccinimide.

Human serum transferrin was fragmented by N-bromosuccinimide and reduction-alkylation. It was observed that there were at least two each of tryptophanyl-serine and tryptophanyl-aspartic acid, and one each of tryptophanyl-alanine and tryptophanyl-glutamic acid bonds. The size of fragments detected by polyacrylamide gel electrophoresis ranged from 8,000 to 70,000 daltons. Several of the fragments were isolated in a homogeneous form with respect to molecular weight, but were shown to be mixtures of at least five molecular species each by end group analysis.

Alanine↗
PubMed1975

N-bromosuccinimide oxidation of anti-DNP and anti-DNP-p-aminobenzoylglutamate antibodies in the presence and absence of protecting hapten.

The reagent N-bromosuccinimide (NBS) has been employed to investigate the role of tryptophan in hapten binding in anti-DNP (H-1) and anti-DNP-p-aminobenzoylglutamate (DNP-ABG) (I-13) antibodies. In 0-1 M acetate (pH 4-0) buffer fifteen and sixteen moles of tryptophan in the anti-DNP and anti-DNP-ABG antibodies respectively were reactive toward NBS. The hapten DNP-lysine protected 1 tryptophan in antibody H-1 and three tryptophans in antibody I-13 from NBS modification. DNP-ABG protected three tryptophans in antibody H-1 and five tryptophans in antibody I-13 from NBS oxidation. NBS treatment of the unprotected antibodies resulted in a significant, but not total inhibition of hapten binding, while in the hapten protected antibody preparation no significant loss of binding occurred due to NBS treatment. The binding activity of the anti-DNP antibody H-1 was more sensitive to NBS oxidation than was the anti-DNAP-ABG antibody I-13. This was apparently due to the larger number of oxidizable tryptophans in I-13 making it less sensitive to overall tryptophan modification. The results of these investigations are discussed in terms of the antibody combining site model proposed by Haselkorn et al. (Haselkorn, Friedman, Givol and Pecht, 1974) derived from kinetic mapping of the antibody-combining site by chemical relaxation spectroscopy.

Aminobenzoates↗
PubMed1976

Structure-activity relationships of some selected beta-adrenergic blocking agents--oxidation with N-bromosuccinimide.

A relationship between in vitro rate of oxidation by N-bromosuccinimide (NBS) and the pharmacologic activity (pA2) of different beta-adrenergic blockers for different blocking agent-tissue combinations has been studied. The rates of oxidation of the alcoholic group in the drugs by NBS, as well as their molecular conformations as represented by molecular models, were studied in order to determine requirements for selectivity and potency of action of beta-adrenergic blocking agents. Using data from all 7 drugs studied--both nonselective and selective blocking agents--no significant correlation between pA2 and -log k2 (k2 is the second order rate constant for the oxidative reaction) was found. If data from only the 4 nonselective agents were used (16 drug-tissue combinations), a correlation significant at p less than 0.01 was found. Hypotheses are presented to account for the selective action of some beta-adrenergic blocking agents.

Adrenergic beta-Antagonists↗
PubMed1977

The reaction of N-bromosuccinimide with enolase.

Ten tryptophan residues per one protein molecule were found to be present in the enolase from human and swine muscle. In Tris buffer, N-bromosuccinimide (NBS) inactivated the enolases after oxidation of all 10 tryptophan residues. The presence of 2-phosphoglycerate (2-PGA) partially protected the activity, and in the presence of 2-PGA together with Mg2+ full protection was observed. In phosphate buffer, only 6 tryptophan residues could be oxidized, but the enzyme was fully inactivated. 2-PGA made possible the oxidation of all 10 tryptophan residues, concomitant with full inactivation. In either case, Mg2+ had no effect. The Km values and pH optima were the same for the native and partially NBS-modified enolases.

Animals↗
PubMed1976

Amino acid sequence of flagellin of Bacillus subtilis 168. III. Tryptic peptides, N-bromosuccinimide peptides, and the complete amino acid sequence.

Of the 28 expected tryptic peptides from Bacillus subtilis 168 flagellin, 24 were isolated and sequenced. Several overlapping tryptic peptides were also characterized. Studies were also performed on two peptides of 142 and 162 residues isolated after cleavage of the flagellin molecule at the single tyrosine residue (residue 142) with N-bromosuccinimide. These studies together with the previous data on the cyanogen bromide peptides and the tryptic peptides from maleylated flagellin permitted the complete amino acid sequence to be established: (see article). The primary structure reveals no obvious regularities or major repetitions of homologous sequences. Hydrophobic residues are distributed randomly in the amino acid sequence. However, the distribution of charged residues is strikingly asymmetric. The NH2-terminal region (residues 1 to 101) possesses a net charge of 6 plus, the middle of the molecule (residues 102 to 203), a net charge of 9 minus, and the COOH terminal region (residues 204 to 304), a net charge of 4 minus.

Amino Acid Sequence↗
PubMed1975

Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c.

N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.

Binding Sites↗