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PubMed1978

Use of ethidium bromide fluorescence enhancement to detect duplex DNA and DNA bacteriophages during zone sedimentation in sucrose gradients: molecular weight of DNA as a function of sedimentation rate.

Duplex DNA molecules and DNA bacteriophages have been sedimented through 5--25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DNAs from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0.38 when 0.025--0.70 microgram of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide--sucrose gradients and detected by fluorescence enhancement.

Centrifugation, Density Gradient↗
PubMed1977

Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids.

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

Chemical Phenomena↗
PubMed1976

Circular dichroism and ethidium bromide binding capacity of chromatin from cells temperature sensitive for the transformed phenotype.

Clone H6-15/163 is a clone of cells, originally derived from SV-40 transformed 3T3 cells, which express the transformed phenotype at low (32 degrees C) but not at high (39 degrees C) temperature. Chromatin was isolated from these cells grown at either temperature and studied by circular dichroism and for its ability to bind the intercalating dye, ethidium bromide. During the exponential phase of growth the chromatins of cells grown at either 32 or 39 degrees C are undistinguishable. Cessation of growth in confluent cultures results in marked changes in circular dichroism spectra and in ethidium bromide binding capacity of chromatin. The changes are much are much more pronounced at 39 degrees C (where the cells truly become quiescent) than at 32 degrees C (where cell proliferation continues although the number of cells per culture remains stationary). Temperature shifts and medium replacement also cause changes in chromatin structure, but the changes are again related to the extent of cell proliferation. It is concluded that the chromatin changes occurring in H6-15/163 cells and detectable by circular dichroism and ethidium bromide binding can be related to the proliferating activity of the cultured cells rather than to the expression of the transformed or untransformed phenotype.

Binding Sites↗
PubMed1976

Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin.

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.

Acetophenones↗
PubMed2004

A comparison of structure and stability between the group 11 halide tetramers M4X4 (M = Cu, Ag, or Au; X = F, Cl, Br, or I) and the group 11 chloride and bromide phosphanes (XMPH3)4.

The tetramers of the group 11 (I) halides, M(4)X(4) (M = Cu, Ag, or Au; X = F, Cl, Br, or I), and corresponding group 11 (I) phosphanes, chloride and bromide (XMPH(3))(4) (X = Cl or Br), are investigated by the density functional theory. All coinage metal(I) halide tetramers adopt squarelike ring structures with an out-of-plane distorted (butterfly) D(2d) symmetry. These structures are much lower in energy than the more compact cubelike T(d) arrangements, which maximize dipole-dipole interactions and more closely resemble the solid-state structures of the copper and silver halides. Phosphine coordination completely changes the structures of these M(4)X(4) clusters. The copper(I) and silver(I) phosphane chloride and bromide tetramers adopt a heterocubane structure, slightly preferred over a step (ladder-type)-cluster structure well-known in the coordination chemistry of such compounds. In stark contrast, gold(I) phosphane chloride and bromide tetramers prefer assemblies of linear XAuPH(3) units with direct gold-gold contacts, resulting in a square planar, centered trigonal planar, or tetrahedral gold core.

Journal Article↗
PubMed1996

Structural Chemistry of Poly(ethylene glycol) Complexes of Lead(II) Nitrate and Lead(II) Bromide.

The reaction of 1:1 stoichiometries (1:1.5 for the nitrate/tetraethylene glycol (EO4) and pentaethylene glycol (EO5) complexes) of PbX(2) (X = NO(3), Br) with five- to eight-donor poly(ethylene glycols) (PEGs) in 3:1 CH(3)CN/CH(3)OH (CH(3)CN only for the nitrate/EO5 complex) followed by solvent evaporation resulted in six crystalline materials upon which X-ray structural analyses were carried out: [Pb(NO(3))(2)(EO4)](n)(), [Pb(NO(3))(2)(EO5)], [Pb(NO(3))(2)(EO6)], [PbBr(EO5)(&mgr;-Br)PbBr(2)].H(2)O, [PbBr(NCMe)(EO6)](2)[PbBr(2)(EO6)][PbBr(3)](2), and [PbBr(EO7)][PbBr(3)]. The nitrates crystallize as tight ion pairs with the PEG ligands coordinating in an equatorial plane around the Pb(2+) ions. Because EO4 has only five oxygen donors, this complex exhibits steric unsaturation which is overcome by a monodentate interaction with a third nitrate anion that is also coordinated to a neighboring Pb(2+) ion. The six donors of EO5 coordinate in an equatorial plane resulting in a 10-coordinate complex with trans, twisted, bidentate nitrate anions. The seven-donor hexaethylene glycol (EO6) only uses six of its oxygen donors to coordinate Pb(2+). [Pb(NO(3))(2)(EO4)](n)() is monoclinic, P2(1)/c, with a = 7.902(3) Å, b = 22.136(6) Å, c = 8.910(2) Å, beta = 90.96(3) degrees, and Z = 4. [Pb(NO(3))(2)(EO5)] is triclinic P&onemacr;, with a = 9.332(3) Å, b = 10.025(3) Å, c = 11.688(4) Å, alpha = 68.41(3) degrees, beta = 68.39(3) degrees, gamma = 68.58(3) degrees, and Z = 2. [Pb(NO(3))(2)(EO6)] is monoclinic P2(1)/c, with a = 16.289(4) Å, b = 10.773(4) Å, c = 12.329(4) Å, beta = 106.77(2) degrees, and Z = 4. Lead(II) bromide complexes with PEGs tend to crystallize as PEG complexed cations with polymeric lead(II) bromide anions. In the EO5 complex, bromide anions in the polymer also coordinate to the PEG-wrapped Pb(2+) cations. The hexa- and heptaethylene glycol (EO6 and EO7, respectively) complexes contain discreet ions. In these halide complexes, EO7 is the only PEG to expand the Pb(2+) coordination number from eight to nine. [PbBr(EO5)(&mgr;-Br)PbBr(2)].H(2)O is triclinic P&onemacr;, with a = 7.922(6) Å,b = 15.802(9) Å, c = 19.001(9) Å, alpha = 73.19(8) degrees, beta = 88.91(9) degrees, gamma = 87.22(9) degrees, and Z = 4. [PbBr(NCMe)(EO6)](2)[PbBr(2)(EO6)][PbBr(3)](2) is monoclinic P2(1)/c, with a = 14.389(4) Å, b = 31.931(9) Å, c = 8.029(2) Å, beta = 97.76(3) degrees, and Z = 2. [PbBr(EO7)][PbBr(3)] is monoclinic Cc, with a = 13.165(3) Å, b = 24.732(5) Å, c = 8.007(1) Å, beta = 94.58(2) degrees, and Z = 4.

Journal Article↗
PubMed2006

Five-coordinate aluminum bromides: synthesis, structure, cation formation, and cleavage of phosphate ester bonds.

The alkane elimination reaction between Salen((t)Bu)H(2) ligands and diethylaluminum bromide was used to prepare three Salen aluminum bromide compounds salen((t)Bu)AlBr (1) (salen = N,N'-ethylenebis(3,5-di-tert-butylsalicylideneimine)), salpen((t)Bu)AlBr (2) (salpen = N,N'-propylenebis(3,5-di-tert-butylsalicylideneimine)), and salophen((t)Bu)AlBr (3) (salophen = N,N'-o-phenylenenebis(3,5-di-tert-butylsalicylideneimine)). The compounds contain five-coordinate aluminum either in a distorted square pyramidal or a trigonal bipyramidal environment. The bromide group in these compounds could be displaced by triphenylphosphine oxide or triphenyl phosphate to produce the six-coordinate cationic aluminum compounds [salen((t)Bu)Al(Ph(3)PO)(2)]Br (4), [salpen((t)Bu)Al(Ph(3)PO)(2)]Br (5), [salophen((t)Bu)Al(Ph(3)PO)(2)]Br (6), and [salophen((t)Bu)Al[(PhO)(3)PO)](2)]Br (7). All the compounds were characterized by (1)H, (13)C, (27)Al, and (31)P NMR, IR, mass spectrometry, and melting point. Furthermore, compounds 1-3 and 5-7 were structurally characterized by single-crystal X-ray diffraction. Compounds 1-3 dealkylated a series of organophosphates in stoichiometric reactions by breaking the ester C-O bond. Also, they were catalytic in the dealkylation reaction between trimethyl phosphate and added boron tribromide.

Journal Article↗
PubMed2000

The hydroboration of propargyl bromide. Simple one-Pot three-component routes to (Z)-1-bromoalk-1-en-4-ols and to anti-homoallylic alcohols.

The hydroboration of propargyl bromide with dialkylboranes takes place regioselectively to give 3-bromoprop-1-en-1-yl dialkylboranes 13 which, upon quaternization with bromide ion, undergo a series of transformations into a number of allylic boron species. By a suitable choice of the experimental conditions it is possible to trap the reaction intermediates with aldehydes and to steer the process toward either the synthesis of (Z)-1-bromoalk-1-en-4-ols 6 or anti-homoallylic alcohols 8. Two one-pot three-component processes were developed based on a sequence of four reactions; preparation of dialkylborane and hydroboration of propargyl bromide are the first steps. Then, quaternization with TEBABr may be carried out either in the presence of the aldehyde when (Z)-1-bromoalk-1-en-4-ols 6 are requested, or in the absence of the aldehyde in order to allow the formation of gamma-substituted allyl borane 18 which, successively, adds to the aldehyde affording anti-homoallylic alcohols 8.

Journal Article↗
PubMed2002

Dimethylformamide as a carbon monoxide source in fast palladium-catalyzed aminocarbonylations of aryl bromides.

Dimethylformamide (DMF) acts as an efficient source of carbon monoxide and dimethylamine in the palladium-catalyzed aminocarbonylation (Heck carbonylation) of p-tolyl bromide to provide the dimethylamide. Addition of amines to the reaction mixture in excess delivers the corresponding aryl amides in good yields. The amines employed, benzylamine, morpholine, and aniline, all constitute good reaction partners. The reaction proceeds smoothly with bromobenzene and more electron-rich aryl bromides, but electron-deficient aryl bromides fail to undergo aminocarbonylation. The reactions are conducted at 180-190 degrees C for 15-20 min with microwave heating in a reaction mixture containing imidazole and potassium tert-butoxide: the latter is required to promote decomposition of the DMF solvent at a suitable rate. The beneficial effects of controlled microwave irradiation as an energy source for the rapid heating of the carbonylation reaction mixture are demonstrated. The carbonylation procedure reported herein, which relies on the in situ generation of carbon monoxide, serves as a convenient alternative to other carbonylation methods and is particularly applicable to small-scale reactions where short reaction times are desired and the direct use of carbon monoxide gas is impractical.

Journal Article↗
PubMed2003

Rapid, easy cyanation of aryl bromides and chlorides using nickel salts in conjunction with microwave promotion.

We report here a fast, easy, and efficient method for the preparation of aryl nitriles from aryl bromides and chlorides. The methodology for aryl bromides involves the use of either Ni(CN)(2) or NaCN and NiBr(2). With aryl chlorides, a mix of NaCN and NiBr(2) is used and the reaction proceeds via the in situ formation of the corresponding aryl bromide. The reaction can be performed in air and is complete within 10 min.

Journal Article↗
PubMed2006

Indium(I) iodide promoted cleavage of diphenyl diselenide and disulfide and subsequent palladium(0)-catalyzed condensation with vinylic bromides. A simple one-pot synthesis of vinylic selenides and sulfides.

[reaction: see text] Diphenyl diselenide (and disulfide) undergo facile reaction with indium(I) iodide and the corresponding intermediate complex condenses in situ with a variety of substituted vinyl bromides in the presence of a catalytic amount of tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] in THF at room temperature to produce vinylic selenides and sulfides in good yields. The conversion of (E)-vinyl bromides is remarkably stereoselective giving (E)-vinyl selenides (and sulpfides) whereas the stereoselectivity in reaction of (Z)-vinyl bromides is not very good.

Journal Article↗
PubMed1997

Formation of Bromocarbenium Bromide Ion Pairs in the Electrophilic Bromination of Highly Reactive Olefins in Chlorinated Aprotic Solvents.

The kinetics and the products of bromination of several substituted stilbenes with tetrabutylammonium tribromide (TBAT) have been investigated in aprotic solvents at different temperatures. Stilbenes bearing electron-withdrawing or moderately electron-donating substituents gave stereospecifically the anti addition products. The reactions followed a second-order rate law, and an inverse kinetic isotope effect (KIE), k(H)/k(D) = 0.85(0.05), was found for the bromination of cis-stilbene. The reactions of cis- and trans-4,4-dimethoxystilbenes yielded mixtures of meso and d,l dibromides both in chloroform and 1,2-dichloroethane. The rate constants (k(Br)()3()-) measured for the latter olefins deviated considerably from the Hammett correlations, and added bromide had a significant effect on the rates. The reactions of these activated stilbenes with molecular Br(2), carried out at low Br(2) concentration, followed a mixed second/third-order rate law. The kinetic and product distribution data for the reaction, with TBAT, of stilbenes bearing electron-withdrawing or moderately electron-donating substituents are interpreted on the basis of the known mechanism involving a product- and rate-determining nucleophilic attack by bromide on the olefin-Br(2) pi-complex. The data related to the bromination of the more activated methoxystilbenes are rationalized considering that, for these olefins, even in aprotic solvents, the ionization of the initially formed 1:1 pi-complex to a bromocarbenium bromide ion pair can compete both with the formation of a bromonium-tribromide ion pair and with the nucleophilic attack by Br(-). For this second-order process (first order in Br(2)), the kinetic constants and the activation parameters have been measured in chloroform and 1,2-dichloroethane and the activation parameters have been compared with those related to the third-order Br(2) addition and to the reaction with TBAT.

Journal Article↗
PubMed2000

Correlation of the rates of solvolysis of cyclopropylcarbinyl and cyclobutyl bromides using the extended Grunwald-Winstein equation

The reactions of cyclopropylcarbinyl bromide (1) and cyclobutyl bromide (2) in hydroxylic solvents proceed with both solvolysis and rearrangement. Depending on the solvent, the reactions of 1 are 10-120 times faster than those of 2, and both are faster than the previously studied allylcarbinyl bromide (3). Specific rates are reported for the reactions of 2 proceeding to solvolysis products and 3. Reactions of 1 proceed to solvolysis products and both 2 and 3; since 2 slowly undergoes further solvolysis, specific rates are obtained by a modified Guggenheim treatment. The two sets of specific rates are analyzed using the extended Grunwald-Winstein equation to give sensitivities toward changes in solvent nucleophilicity of 0.42 for 1 and 0.53 for 2 and corresponding sensitivities toward changes in solvent ionizing power of 0.75 and 0.94. A mechanism is proposed involving a rate-determining ionization with an appreciable nucleophilic solvation of the incipient carbocation.

Journal Article↗
PubMed2005

Characterization of the aggregates of N-alkyl-N-methylpyrrolidinium bromide surfactants in aqueous solution.

We have characterized a new class of surfactant molecules using fluorescence spectroscopic and light-scattering techniques. Our results suggest that this homologous series of N-alkyl-N-methlypyrrolidinium bromide (CnMPB) surfactants with n = 10, 12, 14, 16, and 18 represents a bridge between the well-characterized alkyltrimethylammonium bromide (CnTAB) and dialkyldimethylammonium bromide (di-CnDAB) surfactant series. For the smaller members of the CnMPB series with n = 10, 12, and 14, our results are consistent with the formation of spherical micelles as the surfactant concentration is increased. With increasing alkyl chain length, we observe that the critical micelle concentration decreases and the aggregation number increases, typical of single-tail surfactants. For C16MPB, the formation of micelles at dilute concentrations (0.10 mM) is likely, followed by the coexistence of micelles and small unilamellar vesicles at higher concentrations up to 0.82 mM where only vesicles are present. For C18MPB, our data are consistent with the formation of vesicles only. We demonstrate in this study that the combination of spectroscopic and light-scattering methods is a powerful approach to reveal aspects of aggregate structure and morphology in aqueous CnMPB surfactant systems. In particular, the sensitivity of the fluorescence probe prodan to the polarity of its microenvironment enables the rich complexity of surfactant aggregates exhibited by this series of amphiphilic molecules to be detected.

Journal Article↗
PubMed2004

Efficient aqueous-phase Heck and Suzuki couplings of aryl bromides using tri(4,6-dimethyl-3- sulfonatophenyl)phosphine trisodium salt (TXPTS).

[reaction: see text] Sterically demanding, sulfonated arylphosphines TXPTS and TMAPTS have been applied to the aqueous-phase Heck and Suzuki coupling of aryl bromides. TXPTS provides good yields of Heck coupling products from aryl bromides at 80 degrees C, while both TMAPTS and TPPTS gave significantly less active catalysts. TXPTS is the first ligand to promote the aqueous-phase Heck coupling under such mild conditions. Both TXPTS and TMAPTS provide active catalysts for Suzuki couplings of aryl bromides at 50 degrees C.

Journal Article↗
PubMed2002

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.

Acetylcholine↗
PubMed2004

Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.

The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.

Amino Acid Sequence↗
PubMed1969

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments.

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Amino Acids↗