Long-Lifetime Ru(II) Complexes as Labeling Reagents for Sulfhydryl Groups

Castellano, Felix N.; Dattelbaum, Jonathan D.; Lakowicz, Joseph R.

doi:10.1006/abio.1997.2468

Cited by 90 publications

(92 citation statements)

References 26 publications

(25 reference statements)

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“…This complex shows a maximal anisotropy of 0.33, which is easily sufficient for measurement of the time-resolved anisotropies. In other studies we have found [73,74] that all the rhenium complexes we examined displayed highly polarized emission. It seems that the presence of a single chromophoric group, the diimine ligand, insures a defined direction for the electronic transition and a high fundamental anisotropy.…”

Section: Microsecond Biopolymer Dynamicssupporting

confidence: 55%

“…This property provides information about rotational motions from the steady-state or time-resolved anisotropies. We found that the ruthenium complexes display polarized emission, allowing their use as anisotropy probes [63][64][65][66][67]. For these complexes the emission is from a metal-ligand charge transfer (MLCT) state.…”

Section: Microsecond Biopolymer Dynamicsmentioning

confidence: 98%

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Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

et al. 1999

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The technology and applications of fluorescence spectroscopy are rapidly advancing. In this overview presentation we summarize some recent developments from this laboratory. Two and three-photon excitation have been observed for a wide variety of intrinsic and extrinsic fluorophores, including tryptophan, tyrosine, DNA stains, membrane probes, and even alkanes. It has been possible to observe multi-photon excitation of biopolymers without obvious photochemical or photo-thermal effects. Although not described in our lecture, another area of increasing interest is the use of engineered proteins for chemical and clinical sensing. We show results for the glucose-galactose binding protein from E. coli. The labeled protein shows spectral changes in response to micromolar concentrations of glucose. This protein was used with a novel sensing method based on the modulated emission of the labeled proteins and a long lifetime reference fluorophore. And finally, we describe a recently developed rhenium complex which displays a lifetime near 3 µs in oxygenated aqueous solution. Such long lifetime probes allow detection of microsecond dynamic processes, bypassing the usual nanosecond timescale limit of fluorescence. The result of these developments in protein engineering, sensing methods, and metal-ligand probe chemistry will be the increased use of fluorescence in clinical chemistry and point-of-care analyses.

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Section: Microsecond Biopolymer Dynamicssupporting

confidence: 55%

Section: Microsecond Biopolymer Dynamicsmentioning

confidence: 98%

Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

et al. 1999

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“…Moreover, the fluorescence lifetime of the complex is shorter than the rotational time. To circumvent this difficulty and expand the time window, extrinsic probes with very long lifetime (~800 ns) were used linked to HSA [38] or phosphorescence depolarization methods [39] were applied which contributed to a more accurate value for the rotational time of BSA ca. 42 ns.…”

Section: Resultsmentioning

confidence: 99%

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Andrade¹,

Costa²,

Borst

et al. 2008

J Fluoresc

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The interaction between a free-base, anionic watersoluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a biexponential decay is obtained where a short rotational correlation time (7 int =1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin Jaggregates is induced, which can be detected by timeresolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.

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“…Ruthenium(II) bisbipyridine 5-acetamido-1,10-phenanthroline (½RuðbpyÞ 2 ðIA-phenÞ 2þ ) was synthesized according to the published procedure (22) with the following modification: In lieu of final precipitation as the PF 6 salt, the compound was redissolved in water without further purification. Approximately twofold excess of ½RuðbpyÞ 2 ðIA-phenÞ 2þ was added to a 20 μM solution of C62A/C156S/K97C P450-BM3 in 20 mM Tris buffer (pH 8), and shaken in the dark at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Photooxidation of cytochrome P450-BM3

Ener

Lee

Winkler

et al. 2010

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

109

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High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru II photosensitizer to a nonnative cysteine near the heme (Ru II K97C -Fe III P450 ), in order to promote electron transfer from the Fe III porphyrin to photogenerated Ru III . The Ru II K97C -Fe III P450 conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an Fe IV -hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.ruthenium bipyridine | enzyme catalysis T he cytochromes P450 constitute a superfamily of thiolateligated heme enzymes so named because the Soret absorption band in their CO-bound derivatives peaks near 450 nm. These monooxygenases catalyze a dazzling array of regio-and stereospecific oxidation reactions, including the hydroxylation of aliphatic and aromatic hydrocarbons and the epoxidation of alkenes (1, 2). P450s take two reducing equivalents from NAD(P)H and deliver one atom from dioxygen to the organic substrate; the other oxygen atom is released as water. The consensus mechanism for P450 catalysis (Fig. 1) implicates a ferryl porphyrin radical cation [compound I (CI): Fig. 1, intermediate 6] as the active oxygenating agent (3), but this elusive species has not been observed in P450 under single-turnover or steady-state catalytic conditions. In the postulated mechanism, CI is proposed to abstract a hydrogen atom from the substrate to form transient Fe IV -hydroxide complex (compound II, CII), followed by radical recombination to produce oxygenated product (4, 5). Mechanistic studies of P450 catalysis in cryogenic matrices have suggested that the barrier to formation of CI (5 → 6) is higher than that for its reaction with substrate (6 → 7 → 1) (6). Consequently, recent efforts have focused on alternate routes to P450 CI that bypass the hydroperoxide intermediate (5). One approach employs generation of CII using peroxynitrite, followed by laser flash photolysis to yield CI (7). This technique has been used in studies of the spectra and reactivity of the putative CI species, but the interpretation of the results remains open to question.In earlier work, we employed ½RuðbpyÞ 3 2þ (bpy ¼ 2;2 0 -bipyridine) in a bimolecular flash-quench photochemical oxidation protocol to generate CII and CI in horseradish peroxidase (HRP) and the heme octapeptide from cyctochrome c (MP8) (8, 9). This approach was uns...

show abstract

Long-Lifetime Ru(II) Complexes as Labeling Reagents for Sulfhydryl Groups

Cited by 90 publications

References 26 publications

Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Photooxidation of cytochrome P450-BM3

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