Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra

Berry, Edward A.; Trumpower, Bernard L.

doi:10.1016/0003-2697(87)90643-9

Cited by 867 publications

(729 citation statements)

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“…To obtain the CO-reduced spectrum, dithionite-reduced samples were incubated with CO gas for 2e3 min before spectral acquisition. The pyridineehemochrome method [39] was used to quantify the type b hemes present in the above mentioned samples.…”

Section: Spectroscopic Techniquesmentioning

confidence: 99%

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

et al. 2011

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a b s t r a c tThe organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species.Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo 3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.

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Section: Spectroscopic Techniquesmentioning

confidence: 99%

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

et al. 2011

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“…The concentrations of heme present in the sample can be determined using the pyridine hemochrome absorbance (PHA) method, which relies on the known extinction coefficient for c-type heme in the presence of excess sodium hydroxide and pyridine (e PHA ). 16 This value can be used to calculate the concentration of proteins carrying c-type heme, as it has been shown to be independent of the polypeptide chain. 16 Az-Hm14 and MBP-Hm16 Fe(II) PHA spectra gave the characteristic 550-and 521-nm bands in the visible region, indicating proper incorporation of c-type heme to the tag (Supporting Information Fig.…”

Section: Quantification Of Heme-tagged Proteinsmentioning

confidence: 99%

“…16 This value can be used to calculate the concentration of proteins carrying c-type heme, as it has been shown to be independent of the polypeptide chain. 16 Az-Hm14 and MBP-Hm16 Fe(II) PHA spectra gave the characteristic 550-and 521-nm bands in the visible region, indicating proper incorporation of c-type heme to the tag (Supporting Information Fig. 5).…”

Section: Quantification Of Heme-tagged Proteinsmentioning

confidence: 99%

“…The concentration of protein was calculated using the extinction coefficient for c-type heme in pyridine buffer of 30.27 mM À1 cm À1 at 550 nm as reported by Berry and Trumpower. 16 The average concentration of the reduced PHA samples was used to calculate extinction coefficients at different wavelengths for the spectra of the oxidized samples. Oxidized samples without exogenous ligand to heme were prepared as described above for the PHA samples, except that 920 lL of 50 mM NaP i , pH 7.0 was used instead of the pyridine and dithionite solutions.…”

Section: Purification Of Az-hm14 and Mbp-hm16 Using The His Resinmentioning

confidence: 99%

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A heme fusion tag for protein affinity purification and quantification

Asher

Bren

2010

Protein Science

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We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200-500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.

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“…The molecular mass of RebD was determined using gel filtration by comparison to a set of known standards (Sigma-Aldrich), and by MALDI-TOF mass spectrometry, using BSA as a standard. The heme content of RebD was analyzed using the pyridine hemochrome assay (17), and the iron content was determined using the Ferene S assay (18). Inductively coupled plasma mass spectrometry (ICP-MS) analysis was conducted by Elemental Research Inc. to detect the presence of seven metals in a sample of RebD (Fe, Mn, Co, Ni, Cu, Zn, and Mo); a standard solution containing only buffer was used to correct for any background presence of metals in the buffer solution.…”

Section: Aminomentioning

confidence: 99%

Enzymatic Generation of the Chromopyrrolic Acid Scaffold of Rebeccamycin by the Tandem Action of RebO and RebD

2005

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During the biosynthesis of the fused six-ring indolocarbazole scaffolds of rebeccamycin and staurosporine, two molecules of L-tryptophan are processed to a pyrrole-containing five-ring intermediate known as chromopyrrolic acid. We report here the heterologous expression of RebO and RebD from the rebeccamycin biosynthetic pathway in Escherichia coli, and tandem action of these two enzymes to construct the dicarboxypyrrole ring of chromopyrrolic acid. Chromopyrrolic acid is oxidized by six electrons compared to the starting pair of L-tryptophan molecules. RebO is an L-tryptophan oxidase flavoprotein and RebD a heme protein dimer with both catalase and chromopyrrolic acid synthase activity. Both enzymes require dioxygen as a cosubstrate. RebD on its own is incompetent with L-tryptophan but will convert the imine of indole-3-pyruvate to chromopyrrolic acid. It displays a substrate preference for two molecules of indole-3-pyruvic acid imine, necessitating a net two-electron oxidation to give chromopyrrolic acid.Rebeccamycin 1 and staurosporine 2 ( Figure 1) are indolocarbazole antibiotics originally isolated from the actinomycetes LecheValieria aerocolonigenes and Streptomyces longisporoflaVus, respectively. Rebeccamycin is an inhibitor of DNA topoisomerase I, with a minimum inhibitory concentration of 1.75 µM (1). Staurosporine is one of the strongest inhibitors of protein kinases, displaying an IC 50 of 2.7 nM for protein kinase C (2) and IC 50 's in the range of 1-20 nM for most protein kinases. Because of the importance of both the protein kinases and DNA topoisomerases in cell growth and proliferation, these two compounds have been extensively studied as antitumor drug candidates. Analogues of both rebeccamycin and staurosporine have entered clinical trials for the treatment of neoplastic tumors (3, 4), renal cell cancer (5), and leukemia (6).The fused six-ring indolocarbazole scaffold in 1 and 2 is representative of a variety of natural products and derived from the dimerization of two molecules of L-tryptophan (LTrp) 1 via a complex set of oxidative transformations (7-10) (Scheme 1). Since this natural product scaffold is featured in molecules with an interesting range of biological activities, there has been substantial interest in understanding the enzymology of the oxidative dimerization and ring fusion from the starting pair of L-Trp substrates. Subsequent N-glycosylation of the aglycone through the anomeric carbon of a glucose moiety (in the case of rebeccamycin) and through both C 1 and C 5 of L-ristosamine, the deoxyhexose sugar of staurosporine, followed by the action of methyltransferases (11), leads to the active natural products (Scheme 1). In the case of staurosporine, the enzymatic generation of the N-C linkage between one indole nitrogen and C5 of the aminodeoxyhexose is of notable interest as a novel transformation (11,12).The biosynthetic gene clusters for rebeccamycin and staurosporine have been sequenced and functionally expressed in the heterologous host Escherichia coli, validatin...

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Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra

Cited by 867 publications

References 17 publications

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

A heme fusion tag for protein affinity purification and quantification

Enzymatic Generation of the Chromopyrrolic Acid Scaffold of Rebeccamycin by the Tandem Action of RebO and RebD

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