Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

O’Riordan, Tomás C.; Soini, Aleksi E.; Papkovsky, Dmitri B.

doi:10.1006/abio.2001.4989

Cited by 42 publications

(43 citation statements)

References 23 publications

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“…The monofunctional pentafluorophenyl derivative of PtCPTE was prepared according to a procedure described previously [52]. The luminescent cell viability assay kit CellTiter-Glo was obtained from Promega (Madison, WI, USA), Lipofectamine 2000, MitoTracker Green, LysoTracker Green and Alexa488-transferrin fluorescent probes were obtained from Invitrogen (Bio Sciences Ltd., Dun Laoghaire, Ireland).…”

Section: Methodsmentioning

confidence: 99%

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

et al. 2010

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Research on cell‐penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans‐activator of transcription (TAT)‐derived peptides and, to a lesser extent, on proline‐rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen‐sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15–24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH‐SY5Y and HeLa, via cell‐type‐dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time‐resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen‐sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O2 levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

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Section: Methodsmentioning

confidence: 99%

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

et al. 2010

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“…They emit in the near-infrared with maxima at 767 and 796 nm, respectively. The quantum yields are smaller than for the PtCP probe, while the phosphorescence lifetimes are threefold shorter for PtCPK and twofold longer for PdCPK (2,28,35). The probes are therefore compatible with conventional fluorescent microscopy systems.…”

Section: Design Of Intracellular Oxygen Probes and Their Photophysicamentioning

confidence: 66%

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

O’Riordan¹,

Fitzgerald²,

Ponomarev³

et al. 2007

American Journal of Physiology-Regulatory, Integrative and Comp

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Papkovsky DB. Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging. Am J Physiol Regul Integr Comp Physiol 292: R1613-R1620, 2007. First published December 14, 2006; doi:10.1152/ajpregu.00707.2006.-The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptormediated Ca 2ϩ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer. metalloporphyrin; mitochondrial function; uncoupling MOLECULAR OXYGEN IS A KEY substrate in aerobic biological systems, providing the terminal electron acceptor of the electron transport chain (7). The rate of oxygen consumption is closely regulated by the ATP/ADP ratio (24), while also being highly dependent on the concentration of available oxygen (31) and the action of signaling molecules such as NO (9, 37) and Ca 2ϩ (10, 23), which, in turn, are related to pathways of cell survival and death (22). Furthermore, the induction of cellular hypoxia results in the activation of a specific adaptive transcriptional response governed by the hypoxia inducible transcription factors (9). Oxygen consumption is, therefore, an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. It may be used to elucidate biochemical pathways of the cell and alterations in metabolism caused by various stimuli or disease states. Accordingly, there is a requirement for methods that can effectively monitor cellular oxygen levels and oxygen consumption rates.Optical schemes, based on the quenching by molecular oxygen of long-decay fluorescent and phosphorescent dyes, such as metalloporphyrins and ruthenium(II) complexes, have been under active development in recent years (32). The group of Wilson and coworkers (8, 47) ha...

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“…It is known in the literature that phosphorescence of metalloporphyrins adsorbed on solid surfaces is quenched by atmospheric oxygen only to a minor extent (O'Riordan et al, 2001;Papkovsky et al, 1994). This is in line with our finding, where the most intense phosphorescence emission was observed for the dry uncovered specimens without a mountant and a cover glass.…”

Section: Discussionmentioning

confidence: 98%

“…The conjugate was separated from the free label by gel filtration (Amersham-Pharmacia Sephadex G-50) by using PBS as eluent. The substitution degree of the conjugate was determined photometrically as described in the literature (O'Riordan et al, 2001). The substitution degree was 4.9 labels per mAb.…”

Section: Labeling Of Mab With Platinum(ii) Coproporphyrinmentioning

confidence: 99%

Phosphorescent metalloporphyrins as labels in time‐resolved luminescence microscopy: Effect of mounting on emission intensity

Soini

Sevéus

Meltola

et al. 2002

Microscopy Res & Technique

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In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media.

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Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

Cited by 42 publications

References 23 publications

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

Phosphorescent metalloporphyrins as labels in time‐resolved luminescence microscopy: Effect of mounting on emission intensity

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