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1.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues.  相似文献   

2.
1H and 13C nuclear-magnetic-resonance spectroscopy and functional-group analysis were used to determine the molecular structure of an isolated metabolite (IIb) of trimethyl-lysine as 3-hydroxy-N6-trimethyl-lysine, an important intermediate in the conversion of trimethyl-lysine into trimethylammoniobutyrate and carnitine [Hoppel, Cox & Novak (1980) Biochem. J. 188, 509–519]. Functional-group analysis revealed the presence of a primary amine and reaction of metabolite (IIb) with periodate yielded 4-N-trimethylammoniobutyrate as a product, showing 2,3-substitution on the molecule and suggesting that the 3-substitution on the molecule may be an alcohol ([unk]CH–OH), amine ([unk]CH[unk]–NH2) or carbonyl ([unk]C=O) functional group. 1H integration ratios, 1H and 13C chemical-shift data and 1H and 13C signal multiplicities from the sample (IIb) were used to complete the identification of metabolite (IIb) as 3-hydroxy-N6-trimethyl-lysine. For example, the proton multiplet at δ 4.2p.p.m. and doublet at δ 4.1p.p.m., positions representative of amine or alcohol substitution on methylene carbon atoms, integration ratios of 1:1:2:9:4 and a positive ninhydrin test suggest 3-hydroxy-N6-trimethyl-lysine as the molecular structure for metabolite (IIb). 13C chemical-shift data obtained from the sample (IIb) and compared with several model compounds (trimethylammoniohexanoate, trimethyl-lysine and 3-hydroxylysine) resulted in generation of the spectrum of the metabolite and allowed independent identification of metabolite (IIb) as 3-hydroxy-N6-trimethyl-lysine. The 1H spectrum of erythro- and threo-3-hydroxylysine are presented for comparison, and the 1H and 13C n.m.r. spectra of the erythro-isomer support this analysis.  相似文献   

3.
Phosphonium zwitterions of a known type were obtained in high yield via a 1:1 reaction of p-benzoquinone or methoxy-p-benzoquinone with the tertiary phosphines R3P [R = (CH2)3OH, Ph, Et, Me] and Ph2MeP, in acetone or benzene at room temperature. In all cases, attack of the P-atom occurs at a C-atom rather than at an O-atom. The products were characterized to various degrees by elemental analysis, 31P{1H}, 1H and 13C NMR spectroscopies, and mass spectrometry, and two of the zwitterions, the new [HO(CH2)3]3P+C6H2(O)(OH)(MeO) and the known Ph3P+C6H3(O)(OH), were structurally characterized by X-ray analysis. The PEt3 reaction also produces small amounts of the ‘dimeric’, μ-oxo co-product Et3P+C6H2(O)(OH)-O-C6H3(O)P+Et3 that is tentatively characterized by 1D- and 2D-NMR data. 2,5-Di-tert-butyl- and 2,3,5,6-tetramethyl-p-benzoquinone do not react with [HO(CH2)3]3P under the conditions noted above. Heating D2O solutions of the water-soluble zwitterions R3P+C6H3(O)(OH) [R = (CH2)3OH, Et] at 90 °C for 72 h leads to complete H/D exchange of the H-atom in the position ortho to the phosphonium center.  相似文献   

4.
《Inorganica chimica acta》1988,145(2):273-277
The 13C and 15SN NMR spectra of eleven cis-Fe(S2CNRR′)2(CO)2 complexes, where R and R′ are organic substituents, have been measured at ambient temperature in CDCl3 (0.08–0.16 M). The 13C absorptions for the carbonyl ligands correlate well with the force constants for the CO stretching vibrations in CHCl3 solution. Each of the parameters (13CO absorption and kcis for CO) correlate well with the aqueous solution pKa for+H2NRR′, corrected for the phenyl-containing substituents, high pKa values corresponding to high 13CO absorptions and low kcis CO force constants. [p ]Evidence was found in the 13C NMR spectra for hindered rotation about the CN bond in S2CNC2 in complexes with higher pKa(corr) values and in the 13C spectra of the corresponding thiuram disulfides. [p ]The 15N (natural abundance) NMR spectra for each of the complexes was determined. Each revealed a single sharp absorption in a region of the 15N NMR spectrum which indicates substantial CN double bond character, as one would expect for coordinated dithiocarbamate ligands.  相似文献   

5.
The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are kH,Po = 3 × 106, kCa,Po = 120, kNa,Po = 1, kH,Co = 7 × 104 and kCa,Co = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring kH,Po and kCa,Co which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated ko and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.  相似文献   

6.
Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with 13C at C4′ for use in NMR studies in DMSO-d6 solvent to attempt the detection of a trans-H-bond J-coupling (3hJCCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: 3JH3,OH3 and a smaller, longer range JHH. No evidence for 3hJC4′,OH3 was found. The longer range JHH was traced to 4JH4,OH3 based on 2D 1H–1H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of 4JH2,O3H and 4JH4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.  相似文献   

7.
[17-13C,3H]-Labeled gibberellin A20 (GA20), GA5, and GA1 were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). 13C-Labeled GA29, GA8, GA5, GA1, and 3-epi-GA1, as well as unmetabolized [13C]GA20, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-13C, 3H]GA20 to all three genotypes. 13C-Labeled GA8 and 3-epi-G1, as well as unmetabolized [13C]GA1, were identified by GC-SIM from feeds of [17-13C, 3H]GA1 to all three genotypes. From feeds of [17-13C, 3H]GA5, 13C-labeled GA3 and the GA3-isolactone, as well as unmetabolized [13C]GA5, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-13C, 3H]GA5 to [13C]GA1, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA20, as follows: (a) GA20 → GA1 → GA8; (b) GA20 → GA5 → GA3; and (c) GA20 → GA29. The in vivo biogenesis of GA3 from GA5, as well as the origin of GA5 from GA20, are conclusively established for the first time in a higher plant (maize shoots).  相似文献   

8.
《Inorganica chimica acta》1988,150(1):81-100
The (NH3)5CoOC(NH2)23+ ion is consumed in water according to the rate law k(obs.) = k1 + k2[OH], where k1 = 4.0 × 10−5 s−1 and k2 = 14.2 M−1 s−1 (0–0.1 M [OH];μ = 1.1 M, NaClO4, 25 °C). A hitherto unrecognized intramolecular O- to N- linkage isomerization reaction has been detected. In strongly acid solution only aquation to (NH3)5CoOH23+ is observed, but in 0.1–1.0 M [OH], 7% of the directly formed products is the urea-N complex (NH3)5CoNHCONH22+ which has been isolated. In the neutral pH region a much greater proportion (25%) of the products is the urea-N species. These results are interpreted in terms of an urea-O to urea-N linkage isomerization reaction competing with hydrolysis for both spontaneous (k1) and base-catalyzed (k2) pathways; the rearrangement is not observed in strongly acidic solution (pH ⩽ 1) because the protonated N-bonded isomer (pKa ≈ 3) is unstable with respect to the O-bonded form. The appearance of the isomerization pathway as the pH is raised in the 0–6 region is commensurate with a rate increase which cannot be attributed to a contribution from the base catalysis term k2[OH]. It is argued that this observation establishes, for the spontaneous pathway, that hydrolysis and linkage isomerization are separate reaction pathways — there is no common intermediate. The product distribution and rate data lead to the complete rate law, k(obs.) = k1 + k2[OH] = (ks + kON) + (kOH + kON) [OH] for the reactions of the O-bonded isomers, where ks, kOH are the specific rates for hydrolysis, and kON, kON are the specific rates for O- to N-linkage isomerization, by spontaneous and base-catalyzed pathways respectively; kON = 1.3 × 10−5 s−1 and kON = 1.1 M−1 s−1 (μ = 1.0 M, NaClO4, 25 °C). The O- to N- linkage isomerization has been observed also for complexes of N-methylurea, N,N-dimethylurea and N-phenylurea, but not for the N,N′-dimethylurea species. There is an approximately statistical relationship among the data for −NH2 capture (versus H2O), while −NHR and −NR2 do not compete with water as nucleophiles for Co(III) in either the spontaneous or base-catalyzed hydrolysis processes. For each urea-O complex, O- to N-isomerization is a more significant parallel reaction in the spontaneous as opposed to the base-catalyzed pathway. This is interpreted as being indicative of more associative character in the spontaneous route to products, a conclusion supported by other evidence. Some activation parameter data have been recorded and the effect of the N-substitution on the rates of solvolysis (H2O, Me2SO) is discussed. The urea-N complexes have been isolated as their deprotonated forms, [(NH3)5CoNHCONRR′](ClO4)2·xH2O (R,R′ = H, CH3). They are kinetically inert in neutral to basic solution but in acid they protonate (H2O, pKa 2–3; μ = 1.0 M, 25 °C) and then isomerize rapidly back to their O-bonded forms. Some solvolysis accompanies this N- to O-rearrangement in H2O and Me2SO. Specific rates and activation parameters are reported. The kinetic data follow a rate law of the form kNO(obs.) = (k + kNO)[H+]/(Ka + [H+]) and the active species in the reaction is the protonated form; k, kNO are the specific rates for hydrolysis and isomerization, respectively. Proton NMR data establish that the site of protonation (in Me2SO) is the cobalt-bound nitrogen atom. For the unsubstituted urea species (NH3)5CoNH2CONH23+, diastereotopic exo-NH2 protons arising from restricted rotation about the CN bond are observed. The relevance to the mechanism of the linkage isomerization process is considered. 13C and 1H NMR and electronic absorption spectral data are presented, and distinctions between linkage isomers and the solution structures (electronic and conformational) are discussed. The urea-N/urea-O complex equilibrium is governed by the relation KNO(obs.) = KNO[H+]/[H+](Ka), where KNO is the equilibrium constant = [(NH35Co(urea-O)3+]/[(NH3)5Co(urea-N)3+]. Values for KNO(=kNO/kON = 260 and pKa ≈ 3 for the NH2CONH2 system are consistent with the stability of the N-isomer in feebly acidic to basic solution (e.g. pH 6, KNO(obs.) = 2.6 × 10−2) and instability in acid solution (e.g. pH 1, KNO(obs.) = 240). The equilibrium data for this and other urea complexes of (NH3)5Co(III) are contrasted with the result for the analogous Rh(III)NH2CONH2 system KNO ≈ 1).  相似文献   

9.
《Inorganica chimica acta》1987,134(2):275-278
Silicon-29 (δ 29Si) NMR chemical shifts are reported for the first time for a number of tris(phenyldimethylsilyl)methyl silicon compounds (disilylated derivatives), (PhMe2SiA)3CαL, where LSiBR1R2R3, where R varies widely in electronegativity. δ 29SiB for these series exhibited to some extent good correlations with the electronegativities of the groups bonded to silicon (particularly, δ 29SiBMe2X, X H, OH, OCH3, COCH3 and Cl). Substitution with electronegative atoms shifts the chemical shift of silicon to high field.The 13C NMR spectra of these compounds have been recorded and assigned. The chemical shifts of the α-carbon (Cα) resonances are shown to depend on the type of substituent on the silicon-B, thus 13Cα exhibit downfield shifts when X=oxygen ligand. The 13C phenyl resonances have been measured and show the same order of o, m and p signals, viz. δ ortho (downfield)>δ parameta.The variation of 29Si-1H coupling constants with the electronegativity of X was studied.  相似文献   

10.
Strains pyc13T and ZGT13 were isolated from Lake Pengyan and Lake Zigetang on Tibetan Plateau, respectively. Both strains were Gram-negative, catalase- and oxidase-positive, aerobic, rod-shaped, nonmotile, and nonflagellated bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains pyc13T and ZGT13 belong to the genus Halomonas, with Halomonas alkalicola 56-L4-10aEnT as their closest neighbor, showing 97.4% 16S rRNA gene sequence similarity. The predominant respiratory quinone of both strains was Q-9, with Q-8 as a minor component. The major fatty acids of both strains were C18:1ω6c/C18:1ω7c, C16:1ω6c/C16:1ω7c, C16:0, and C12:0 3OH. The polar lipids of both strains consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, glycolipid, phospholipids of unknown structure containing glucosamine, and unidentified phospholipids. The DNA G + C content of pyc13T and ZGT13 were 62.6 and 63.4 mol%, respectively. The DNA-DNA hybridization values of strain pyc13T were 34, 41, 61, 35, and 35% with the reference strains H. alkalicola 56-L4-10aEnT, H. sediminicola CPS11T, H. mongoliensis Z-7009T, H. ventosae Al12T, and H. fontilapidosi 5CRT, respectively. Phenotypic, biochemical, genotypic, and DNA-DNA hybridization data showed that strains pyc13T and ZGT13 represent a new species within the genus Halomonas, for which the name H. tibetensis sp. nov. is proposed. The type strain is pyc13T (= CGMCC 1.15949T = KCTC 52660T).  相似文献   

11.
The kinetics and mechanism of a linear trihydroxamic acid siderophore (deferriferrioxamine B, H4DFB+) ligand exchange with Al(H2O)63+ to form mono(deferriferrioxamine B)aluminum(III) (Al(H2O)4H3DFB)3+ have been investigated at 25 °C over the [H+] range 0.001−1.0 M and I = 2.0 M (HClO4/NaClO4) by 27Al NMR. Kinetic results are consistent with Al(H2O)4(H3DFB)3+ formation and dissociation proceeding through a parallel path mechanistic scheme involving Al(H2O)63+(k2/k−1) and Al(H2O)5(OH)2+(k2/k−2) where k1 = 0.13 M−1 s−1, k−1 = 8.7 × 10−3 M−1 s−1, k2 = 2.7 × 103 M−1 s−1, and k−2 = 9.6 × 10−4 s−1. Relative complex formation rates at Al(H2O)63+ and Al(H2O)5OH2+, and comparison with kinetic data for a series of synthetic hydroxamic acids, suggest that an interchange mechanism is operative. These results are also discussed in relation to kinetic data for the corresponding iron(III)-deferriferrioxamine B system.  相似文献   

12.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues.  相似文献   

13.
The macrocyclisation reaction of 3,3′-(3,6-dioxaoctane-1,8-diyldioxy)-bis(2-hydroxybenzaldehyde) (1) with S-methylisothiosemicarbazide hydroiodide (H2NNC(SCH3)NH2·HI) in the presence of potassium triflate, followed by addition of M(CH3COO)2·nH2O, where M=Ni, Cu, Zn, afforded [NiLKI3] (2), [NiLK(CF3SO3)] (3), [CuLK(CF3SO3)(CH3OH)] (4) and [(ZnILK)2CH3OH] (5), respectively. Compounds 2-5 have been characterised by X-ray crystallography. IR, electronic, mass, 1H, 13C{1H} and 19F{1H} NMR spectra are reported. Magnetic susceptibility measurements and ESR spectra of 4 indicate weak intermolecular spin-spin interactions, which are mostly dipolar in origin.  相似文献   

14.
Labelled convallatoxin was isolated from leaves of Convallaria majalis after administration of convallatoxol-19-3H and convallatoxol-U-14C, resp. Oxidation of-CH2OH → -CHO at the glycoside level therefore is a possible step in the biogenesis of strophanthidin glycosides.  相似文献   

15.
Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs), 20α-HSDs, as well as the 5β-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3α-HSD (AKR1C9) as a starting structure to engineer either 5β-reductase or 20α-HSD activity. 5β-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a kcat comparable to that for homogeneous rat and human liver 5β-reductases. pH versus kcat profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH2+ character facilitates enolization of the Δ4-3-ketosteroid and subsequent hydride transfer to C5. Since 5β-reductase precedes 3α-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3α-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20α-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log10(kcat/Km) from androgens to progestins was of the order of 1011. This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.  相似文献   

16.
《Inorganica chimica acta》1988,149(2):259-264
The bis(N-alkylsalicylaldiminato)nickel(II) complexes Ni(R-sal)2 with R = CH(CH2OH)CH(OH)Ph (I), R = CH(CH3)CH(OH)Ph (II) and R = CH2CH2Ph (III; Ph = phenyl) were prepared and characterized. In the solid state I and II are paramagnetic (μ = 3.2 and 3.3 BM at 20 °C, respectively), whereas III is diamagnetic. It follows from the UV-Vis spectra that in acetone solution I is six-coordinate octahedral and III is four-coordinate planar, the spectrum of II showing characteristics of both modes of coordination. Vis spectrophotometry and stopped-flow spectrophotometry were applied to study the kinetics of ligand substitution in I–III by H2salen (= N,N′-disalicylidene-ethylenediamine) in the solvent acetone at different temperatures. The kinetics follow a second-order rate law, rate = k[H2-salen] [complex]. At 20 °C the sequence of rate constants is k(III):k(II):k(I) = 11 850:40.6:1. The activation parameters are ΔH(I) = 112, ΔH(II) = 40.7, ΔH(III) = 35.7 kJ mol−1 and ΔS(I) = 92, ΔS(II) = −103, ΔS(III) = −89 J K−1 mol−1. The enormous difference in rate between complexes I, II and III, which is less pronounced in methanol, is attributed to the existence of a fast equilibrium planar ⇌ octahedral, which is established in the case of I and II by intramolecular octahedral coordination through the hydroxyl groups present in the organic group R. An A-mechanism is suggested to control the substitution in the sense that the entering ligand attacks the four-coordinate planar complex, the octahedral complex being kinetically inert.  相似文献   

17.
《Inorganica chimica acta》1987,131(2):241-245
Complexes of 3-hydroxy-2-naphthaldehyde benzylhydrazone (H2nabh) and 3-hydroxy-2-naphthaldehyde salicyloylhydrazone (H3nash) of the empirical composition M(L2H)·nH2O [M = manganese(II), iron(II), cobalt(II), nickel(II), copper(II), zinc(II), cadmium(II), mercury(II), L = H2nabh, H3nash and n = 0, 1, 2] were prepared and characterized by elemental analyses, magnetic susceptibility, electronic and infrared spectral data. Zinc(II) and cadmium(II) complexes were also studied by 13C, 1H NMR and the Cu(nabh)·H2O complex by transmission electron microscopy. The complexes are coloured and highly insoluble in common organic solvents. Absence of the original anion in the complexes indicates deprotonation of the ligands (H2nabh and H3nash) which bind the metal ions from the OH and the CN groups.  相似文献   

18.
Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K m, k cat, and k cat/K m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K m, k cat, and k cat/K m for Ac-nKRR-amc substrate were 100 μM, 0.112 s?1, and 1120 M?1·s?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.  相似文献   

19.
《Inorganica chimica acta》1988,149(1):139-145
The stoichiometry and kinetics of the reaction between [Cu(dien)(OH)]+ and [Fe(CN)6]3− in aqueous alkaline medium are described. The rate equation − (d[Fe(III)]/dt = {k1[OH]2[[Cu(dien)(OH)]+] + k2[OH] × [[Cu(dien)(OH)]+]2}([Fe(III)]/[Fe(II)]) (Fe(III) = [Fe(CN)6]3−; Fe(II) = [Fe(CN)6]4−, the 4:4:1 OH/Fe(III)/[Cu(dien)(OH)]+ stoichiometric ratio and the nature of the ultimate products identified in the reaction solution suggest the fast formation of a doubly deprotonated Cu(III)-diamido complex which slowly undergoes an internal redox process where the ligand is oxidised to the Schiff base H2NCH2CH2NCHCHNH.The [[Cu(dien)(OH)]+]2 term in the rate equation is explained with the formation of a transient μ-hydroxo mixed-valence Cu dimer. A two-electron internal reduction of the Cu(III) complex yielding a Cu(I) intermediate is suggested to account for the presence of monovalent copper in a precipitate which forms at relatively high reactant concentrations and in the absence of dioxygen.  相似文献   

20.
《Inorganica chimica acta》1988,141(2):263-274
Reduction of some N-alkylimines has been achieved with NaBH4 to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their 1H and 13C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl2L2 complexes. The 1H, 13C and 199Hg NMR chemical shifts have been obtained as well as 1J(13CH) and 2J(13CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the 199Hg.  相似文献   

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