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1.
A chemical method for introducing haptens onto DNA probes   总被引:5,自引:0,他引:5  
We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 X 10(-17) mol (1 X 10(6) copies) of HBV DNA in total DNA from human serum.  相似文献   

2.
Labeling of DNA probes with a photoactivatable hapten   总被引:2,自引:0,他引:2  
A photoactivatable reagent for introducing haptens onto DNA probes has been prepared using a commercially available bifunctional linker arm reagent and amino-derivatized 2,4-dinitrophenyl (DNP). The resulting compound (photo-DNP) couples efficiently to DNA using an ordinary sunlamp. Under optimum conditions, about 7-23 DNP molecules per 1000 bases are incorporated into the DNA. Hybridization experiments demonstrate that as little as 1.5 x 10(5) copies of target DNA can be detected by filter hybridization with a photo-DNP-labeled probe and immunochemical detection.  相似文献   

3.
The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.  相似文献   

4.
Characterisation of a highly hydrophobically modified lactate dehydrogenase   总被引:1,自引:0,他引:1  
1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.  相似文献   

5.
The aldehyde groups formed on periodate oxidation of cell surface sialyl residues were used to insert a mitogenic site onto the lymphocyte membrane by attachment of biotin hydrazide or 2,4-dinitrophenyl hydrazine. The biotin- or 2,4-dinitrophenyl-conjugated cells were both agglutinated and stimulated when cultured with avidin or anti-2,4-dinitrophenyl antibody respectively. On the other hand, biotin or DNP-conjugated cells, modified via functional groups on the membrane proteins, were agglutinated but not stimulated when cultured with avidin or anti-DNP antibody respectively. Our results show that the specific interaction of a protein at the periodate oxidation site leads to blastogenesis.  相似文献   

6.
1-Fluoro-2,4-dinitrobenzene can be used to selectively label histidine, tyrosine, and cysteine residues in maleylated proteins. The usefulness of the resulting chromophores for peptide mapping by high-performance liquid chromatography was demonstrated with the lectin from sainfoin (Onobrychis viciifolia). The 2,4-dinitrophenyl (Dnp) label also can be used in a hydrophobic modulation approach as the mobility of a labeled model peptide changes considerably when its Dnp group is removed by thiolysis. Application of the method for checking sequences obtained by DNA or amino acid methods was shown by experiments with Viciae lectins. The probable cleavage site that generates the pea lectin's beta-chain from the alpha-beta precursor was identified and the sequence differences between the lentil and pea lectin beta-chains were examined.  相似文献   

7.
R J Kraus  S J Foster  H E Ganther 《Biochemistry》1983,22(25):5853-5858
A convenient procedure was developed for identifying selenocysteine in selenoproteins by mass spectroscopy, based on formation of the 2,4-dinitrophenyl (DNP) derivative. Pure ovine erythrocyte glutathione peroxidase was reduced with sodium borohydride and reacted with 1-fluoro-2,4-dinitrobenzene at neutral pH under anaerobic conditions in 4 M guanidine. The inactivated enzyme was hydrolyzed with 6 N HCl for 20 h at 110 degrees C under anaerobic conditions. Following extraction of the hydrolysate with benzene, Se-(2,4-dinitrophenyl)selenocysteine in the aqueous phase was separated from non-DNP-amino acids by gel-filtration chromatography and then separated from other water-soluble DNP-amino acids by reversed-phase high-performance liquid chromatography. The Se-(2,4-dinitrophenyl)selenocysteine was converted to Se-methyl-N-(2,4-dinitrophenyl)selenocysteine by the addition of sodium barbital to induce an intramolecular Se leads to N shift (Smiles rearrangement) under anaerobic conditions, in the presence of methyl iodide to trap the liberated selenol group. Following esterification of the product's carboxyl group with methanol and hydrochloric acid, it was subjected to direct probe mass spectroscopy and identified as the methyl ester of Se-methyl-N-(2,4-dinitrophenyl)selenocysteine. This procedure allows selenocysteine to be isolated quite easily as a readily identifiable derivative and has permitted the first identification of a seleno amino acid in a protein by mass spectroscopy.  相似文献   

8.
DNA fragments were labeled with dinitrophenyl (DNP) residues by the reaction with 2,4-dinitrobenzaldehyde in alkaline condition and the labeled DNA was used as a probe for non-radioactive in situ hybridization. DNP-labeled DNA probes for T cell receptor beta chain, c-myc and HTLV-1 were hybridized in situ to mRNA on cell specimens fixed with Carnoy's fixative. DNA-mRNA hybrids were detected immunohistochemically using anti-DNP antibodies. Cytoplasms of adult T cell leukemia cells were stained with varied intensity when these probes were used. More than 70% of cells were positively stained with T cell receptor probe. However, less than 30% of cells were stained with c-myc and HTLV-1 probes. The present study indicates that non-radioactive in situ hybridization can be used for the characterization and classification of leukemia.  相似文献   

9.
We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K(i) of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P(1) position.  相似文献   

10.
A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.  相似文献   

11.
A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.  相似文献   

12.
The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.  相似文献   

13.
Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2′-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2′-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2′-deoxyinosine 9 was prepared from 2′-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-15N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.  相似文献   

14.
Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.  相似文献   

15.
Antisera to thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) have previously been produced in rabbits by immunization with a conjugate having TRH linked to a carrier protein by means of dinitrophenylene (Dnp) moiety. Studies on the specificity of the antisera obtained suggested that the sensitivity of the radioimmunoassay for TRH may be increased substantially by prior conversion of the hormone in to dinitrophenylene derivatives. To test this possibility, several TRH-Dno derivatives were prepared by reaction of TRH with equimolar amounts of 1,5-difluoro-2,4-dinitrobenzene yielding Nim-(5-fluoro-2,4-dinitrophenyl)TRH. This intermediate was reacted with ammonia, histamine, tyramine or N alpha-acetyl-lysine methyl ester (N alpha Ac-Lys-OMe) to yield the respective unsubstituted and N-substituted Nim-(5-amino-2,4-dinitrophenyl)TRH derivatives: TRH-Dnp-NH2, TRH-Dnp-histamine, TRH-Dnp-tyramine and TRH-Dnp-N alpha Ac-Lys-OMe. Nim-(2,4-Dinitrophenyl)TRH was prepared similarly by reaction of TRH with 1-fluoro-2,4-dinitrobenzene. The products were isolated by means of high-performance liquid chromatography (HPLC) and were found to be pure by HPLC and thin-layer chromatography using several solvent systems. TRH-Dnp-histamine and TRH-Dnp-tyramine were labelled with 125I using the chloramine-T method. The labelled products were purified to homogeneity by ion-exchange chromatography on SP-Sephadex and adsorption chromatography on Sephadex LH-20, respectively, and were found by HPLC to be pure.  相似文献   

16.
A new method was developed to monitor specific protein binding reactions with an ATP-labeled ligand and firefly luciferase. The ligand, 2,4-dinitrobenzene, was covalently coupled to four ATP derivatives and three of these conjugates were measured quantitatively at nanomolar levels with firefly luciferase. Incubation of the conjugates with antibody to the 2,4-dinitrophenyl residue diminished the peak light intensities produced in the bioluminescent assay, whereas incubation with immunoglobulin from a nonimmunized rabbit did not affect light production. Therefore, the antibody-bound ligand-ATP conjugates were inactive in the bioluminescent assay and levels of unbound conjugate could be measured in the presence of the bound form. The firefly luciferase was used to monitor competitive binding reactions between the antibody, the conjugates, and N(2,4-dinitrophenyl)-β-alanine.  相似文献   

17.
A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.  相似文献   

18.
In the cytidine and adenosine derivatives an isomerization of a 2,4-dinitrophenyl group between the 2′- and 3′-positions of the ribose was observed under neutral conditions. Moreover, it was shown that isomerization of the 2,4-dinitrophenyl group in conditions required to synthesize phosphoramidites and lability in aqueous ammonia make chemical synthesis of 2′-O-(2,4-dinitrophenyl) oligonucleotides impossible.  相似文献   

19.
The interaction of chemicals with DNA may lead to genotoxicity, mutation or carcinogenicity. A simple open tubular capillary electrochromatographic method is proposed to rapidly assess the interaction affinity of three environmental contaminants (1,4-phenylenediamine, pyridine and 2,4-diaminotoluene) to DNA by measuring their retention in the capillaries coated with DNA probes. DNA oligonucleotide probes were immobilized on the inner wall of a fused silica capillary that was first derivatized with 3-(aminopropyl)-triethoxysilane (APTES). The difference in retention times and factors was considered as the difference in interaction affinity of the contaminants to the DNA probes. The interaction of the contaminants with both double-stranded (dsDNA) and single-stranded DNA (ssDNA) coatings was compared. Retention factors of 1,4-phenylenediamine, pyridine and 2,4-diaminotoluene in the capillary coated with ssDNA probe were 0.29, 0.42, and 0.44, respectively. A similar trend was observed in the capillary coated with dsDNA, indicating that 2,4-diaminotoluene has the highest affinity among the three contaminants. The relative standard deviation (RSD) for the retention factors was in the range of 0.05–0.69% (n = 3). The results demonstrated that the developed technique could be applied for preliminary screening purpose to provide DNA interaction affinity information of various environmental contaminants.  相似文献   

20.
We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates that permit continuous measurement of the enzyme activity, even in crude tissue extracts.  相似文献   

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