Chelating agents for the binding of metal ions to macromolecules

Sundberg, Michael W.; Meares, Claude F.; Goodwin, David A.; Diamanti, Carol I.

doi:10.1038/250587a0

Cited by 86 publications

(30 citation statements)

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“…To do this, we exploited a novel method that relies on tethering a DNA cleavage agent to a single specific amino acid side chain of a protein (13). The reagent p-bromoacetamidobenzyl-EDTA⅐Fe (FeBABE) has one reactive group facilitating covalent attachment to cysteine side chains, whereas a second group holds a single metal atom in a tight coordination complex (14). Under appropriate conditions, the divalent cation can participate in the generation of hydroxyl radicals, which attack deoxyribose units, resulting in DNA strand scission (15).…”

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confidence: 99%

Organization of Open Complexes at Escherichia coliPromoters

Bown

Owens

Meares

et al. 1999

Journal of Biological Chemistry

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A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of 70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered 70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA⅐Fe (FeBABE). RNA polymerase core reconstituted with tagged derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the ؊10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of with promoter DNA is discussed.Promoter recognition requires sequence-specific contacts by the transcriptional apparatus. At most promoters these contacts are made upstream from the transcription start point. Once the transcriptional apparatus has bound the promoter to form a closed complex, an isomerization event occurs to generate the open complex, forming the single-stranded template required for transcription. The bacterium Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription elongation with subunit composition ␣ 2 ␤␤Ј. Promoter specificity is principally afforded by a separate subunit, , which associates with the core enzyme to give holoenzyme (RNAP) 1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The 70 subunit, encoded by rpoD, is one of several subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth. RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of 70 to make stable contacts with the promoter DNA (1, 2). E. coli promoters contain two very conserved motifs, the Ϫ10 and Ϫ35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended Ϫ10 motif (5). The extended Ϫ10 motif (5Ј-TGXTATAAT-3Ј) can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within the Ϫ35 region (6, 7). Therefore this TGX motif is able to compensate for a poor Ϫ35 hexamer. The TG motif has been shown to be important for promoter activity in several other bacterial species (8 -11). Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of 70 contact the Ϫ10 and Ϫ35 hexamers, respectively (1, 2), whereas the C-terminal domain of the ␣ subunit (␣CTD) contacts the UP element (4). Recen...

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confidence: 99%

Organization of Open Complexes at Escherichia coliPromoters

Bown

Owens

Meares

et al. 1999

Journal of Biological Chemistry

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“…These results provide forceful evidence that the technique of labeling biological molecules with chelates containing radioactive indium has significant applications to nuclear medicine (11,12).…”

Section: Discussionmentioning

confidence: 99%

Covalent attachment of metal chelates to proteins:the stability in vivo and in vitro of the conjugate of albumin with a chelate of 111indium.

Meares¹,

Goodwin²,

Leung³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Human serum albumin has been conjugated to 1-(p-benzenediazonium)(ethylenedinitrilo)tetraacetic acid, a powerful chelating agent, and radioactive "'1indium ions have been added specifically to the chelating groups. The product, with a specific radioactivity of about 1 mCi/mg of protein, was employed as a radiotracer in scintillation scanning studies with human volunteers. Results show that 48 hr after injection, practically all of the label remains attached to albumin. This is confirmed by electrophoresis of serum proteins; 7 days after injection, 85% of the radioactivity in the serum is still in the albumin fraction. These observations agree with in vitro studies of the labeled albumin in human serum, where loss of the metal ion from the chelating group to the protein transferrin amounts to less than 3% after 1 week and less than 5% after 2 weeks. A metal ion may be attached to a biological molecule by means of a "bifunctional" chelating agent capable of forming a covalent bond to the desired molecule. The conjugation of a derivative of EDTA to a large molecule permits the addition of any of a number of metal ions to form a stable chelate. In principle, this can lead to the utilization of the physical properties of various metal ions in a broad range of applications. Of particular importance is the tagging of certain biological molecules with short-lived radionuclides, and the use of these tagged molecules in medical diagnosis (1).A case in point is the use of chelates containing radioisotopes of indium for tracer studies. The intravenous injection of such compounds into human patients often leads to the binding of a large fraction of the radioactive metal by the serum protein transferrin, with the result that the observed behavior of the radionuclide is characteristic of the transferrin-indium complex (2, 3). The rapid action of apo-transferrin in removing trivalent iron from chelates is also well known (4, 5). Before biomolecules labeled with indium chelates can be considered valid radioindicators in vivo, it must be shown that the chelates are thermodynamically stable or kinetically inert to the extent that loss of indium is negligible.Previously, we have studied the metabolism of proteins conjugated with indium chelates by measuring the biological lifetime of the label in mice and by observing the distribution of radioactivity among, serum proteins in rabbits (1, 6). The results of these studies suggested that the protein-chelate conjugates were reasonably stable in vivo, but that as much as 50% of the metal ion might eventually become bound to transferrin and be sequestered in the liver and bone marrow. The present studies were undertaken to provide a detailed account of the behavior of carrier-free amounts of the albumin-chelate conjugate, both in vivo and under physiological conditions in vitro. MATERIALS AND METHODSAll reagents were the purest commercially available products, used without further purification except where noted. Chromatographic columns and materials were obtained from BioRad Lab...

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“…[5][6] Functionalized EDTA as a BFCA was first used for evaluating a scintigraphy, but this metal complex revealed a low stability for a in vivo condition. [7][8][9] Other bifunctionalized cyclic chelating agents based on oligoaza macrocycle derivatives such as MeO-DOTA-NCS (α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), 2B-DOTA-NCS (2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TEPPA (1,4,8,11-tetraazacyclotetradecan-1,4,8,11-tetrapropionic acid), and so on were then introduced and are now commercially available. [10][11][12] The functionalized pendant-arms of oligoaza derivatives can be utilized to conjugate bioactive molecules, 13 and their complexes with metal ions show a better stability than acyclochelating agents.…”

Section: Tcmentioning

confidence: 99%

Synthesis of Bifunctional Chelating Agent Derived from Lysine and its Radiolabeling with^99mTc

Pyun¹,

Choi²,

Hong³

et al. 2009

Bulletin of the Korean Chemical Society

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Chelating agents for the binding of metal ions to macromolecules

Cited by 86 publications

References 3 publications

Organization of Open Complexes at Escherichia coliPromoters

Organization of Open Complexes at Escherichia coliPromoters

Covalent attachment of metal chelates to proteins:the stability in vivo and in vitro of the conjugate of albumin with a chelate of 111indium.

Synthesis of Bifunctional Chelating Agent Derived from Lysine and its Radiolabeling with^99mTc

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Chelating agents for the binding of metal ions to macromolecules

Cited by 86 publications

References 3 publications

Organization of Open Complexes at Escherichia coliPromoters

Organization of Open Complexes at Escherichia coliPromoters

Covalent attachment of metal chelates to proteins:the stability in vivo and in vitro of the conjugate of albumin with a chelate of 111indium.

Synthesis of Bifunctional Chelating Agent Derived from Lysine and its Radiolabeling with99mTc

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Synthesis of Bifunctional Chelating Agent Derived from Lysine and its Radiolabeling with^99mTc