Design and applications of methods for fluorescence detection of iron in biological systems

2002 ◽  
Vol 30 (4) ◽  
pp. 729-732 ◽  
Author(s):  
B. P. Espósito ◽  
W. Breuer ◽  
Z. I. Cabantchik
Keyword(s):  
Metal Binding ◽  
Biological Systems ◽  
Iron Chelator ◽  
Specific Cell ◽  
Iron Binding ◽  
Physiological Conditions ◽  
High Affinity ◽  
Cell Compartments ◽  
Heterogeneous Nature ◽  
Labile Iron

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.

scholarly journals High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin

2007 ◽  
Vol 404 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Ali G. Khan ◽  
Stephen R. Shouldice ◽  
Shane D. Kirby ◽  
Rong-hua Yu ◽  
Leslie W. Tari ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Metal Binding ◽  
Binding Assay ◽  
Rank Order ◽  
Binding Protein ◽  
Iron Binding ◽  
Wild Type ◽  
Mutant Proteins ◽  
High Affinity ◽  
Iron Binding Protein

The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0×1018 M−1. These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.

scholarly journals Influence of fluorophore and linker length on the localization and trafficking of fluorescent sterol probes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jarmila Králová ◽  
Michal Jurášek ◽  
Lucie Mikšátková ◽  
Anna Marešová ◽  
Jan Fähnrich ◽  
...  
Keyword(s):  
Genetic Mutation ◽  
Cell Permeability ◽  
Specific Cell ◽  
Cell Compartments ◽  
Intracellular Compartments ◽  
Resolution Imaging ◽  
Intracellular Labelling ◽  
Kinetics Of Uptake ◽  
Kinetics Of ◽  
Derivatives Of

AbstractFluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.

scholarly journals Analysis of yggX and gshA Mutants Provides Insights into the Labile Iron Pool in Salmonella enterica

2008 ◽  
Vol 190 (23) ◽  
pp. 7608-7613 ◽  
Author(s):  
Michael P. Thorgersen ◽  
Diana M. Downs
Keyword(s):  
Salmonella Enterica ◽  
Iron Chelator ◽  
Fenton Chemistry ◽  
Labile Iron Pool ◽  
Growth Defects ◽  
Labile Iron ◽  
Iron Pool ◽  
Mutant Phenotypes ◽  
Permeable Iron ◽  
Iron Chelator Deferoxamine

ABSTRACT Strains of Salmonella enterica lacking YggX and the cellular reductant glutathione exhibit defects similar to those resulting from iron deficiency and oxidative stress. Mutant strains are sensitive to hydrogen peroxide and superoxide, deregulate the expression of the Fur-regulated gene entB, and fail to grow on succinate medium. Suppression of some yggX gshA mutant phenotypes by the cell-permeable iron chelator deferoxamine allowed the conclusion that increased levels of cellular Fenton chemistry played a role in the growth defects. The data presented are consistent with a scenario in which glutathione acts as a physiological chelator of the labile iron pool and in which YggX acts upstream of the labile iron pool by preventing superoxide toxicity.

A consensus zinc finger peptide: design, high-affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant

1991 ◽  
Vol 113 (12) ◽  
pp. 4518-4523 ◽  
Author(s):  
Beth Allyn Krizek ◽  
Barbara T. Amann ◽  
Valda J. Kilfoil ◽  
Denise L. Merkle ◽  
Jeremy M. Berg
Keyword(s):  
Zinc Finger ◽  
Metal Binding ◽  
Sequence Variant ◽  
Peptide Design ◽  
High Affinity ◽  
Ph Dependent ◽  
Zinc Finger Peptide

scholarly journals Metal binding spectrum and model structure of theBacillus anthracisvirulence determinant MntA

Metallomics ◽  
2015 ◽  
Vol 7 (10) ◽  
pp. 1407-1419 ◽  
Author(s):  
Elena Vigonsky ◽  
Inbar Fish ◽  
Nurit Livnat-Levanon ◽  
Elena Ovcharenko ◽  
Nir Ben-Tal ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Metal Binding ◽  
Model Structure ◽  
High Affinity ◽  
Virulence Determinant ◽  
System P

TheBacillus anthracisvirulence determinant MntA is a high-affinity manganese system.

The Cellular Labile Iron Pool and Intracellular Ferritin in K562 Cells

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2128-2134 ◽  
Author(s):  
Abraham M. Konijn ◽  
Hava Glickstein ◽  
Boris Vaisman ◽  
Esther G. Meyron-Holtz ◽  
Itzchak N. Slotki ◽  
...  
Keyword(s):  
Biological Effects ◽  
Ferritin Level ◽  
Regulatory Protein ◽  
Iron Chelation ◽  
K562 Cells ◽  
Iron Chelator ◽  
Labile Iron Pool ◽  
Cellular Iron ◽  
Labile Iron ◽  
Iron Pool

Abstract The labile iron pool (LIP) harbors the metabolically active and regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels. Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 μmol/L, as monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator-free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to reappear was relatively small and occurred within 1 hour, whereas the second was larger and relatively slow to occur, paralleling the decline in intracellular ferritin level (t½= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation. The changes in the LIP were also inversely reflected in the activity of iron regulatory protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively similarly in response to long-term treatments with the iron chelator deferoxamine, although L-ferritin declined more rapidly, resulting in a 4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not H-ferritin, was partially attenuated by the lysosomotrophic agent, chloroquine; on the other hand, antiproteases inhibited the degradation of both subunits to the same extent. These findings indicate that, after acute LIP depletion with fast-acting chelators, iron can be mobilized into the LIP from intracellular sources. The underlying mechanisms can be kinetically analyzed into components associated with fast release from accessible cellular sources and slow release from cytosolic ferritin via proteolysis. Because these iron forms are known to be redox-active, our studies are important for understanding the biological effects of cellular iron chelation.

scholarly journals Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

2012 ◽  
Vol 443 (1) ◽  
pp. 307-315 ◽  
Author(s):  
Linda Troeberg ◽  
Barbara Mulloy ◽  
Peter Ghosh ◽  
Meng-Huee Lee ◽  
Gillian Murphy ◽  
...  
Keyword(s):  
Sulfated Polysaccharide ◽  
Pentosan Polysulfate ◽  
Endogenous Inhibitor ◽  
Binding Ability ◽  
Physiological Conditions ◽  
Interaction Sites ◽  
High Affinity ◽  
Multiple Protein ◽  
Trimolecular Complex ◽  
Protein Interaction Sites

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

scholarly journals A Novel Star Like Eight-Arm Polyethylene Glycol-Deferoxamine Conjugate for Iron Overload Therapy

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 329
Author(s):  
Bohong Yu ◽  
Yinxian Yang ◽  
Qi Liu ◽  
Aiyan Zhan ◽  
Yang Yang ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Iron Overload ◽  
Iron Chelator ◽  
Iron Chelators ◽  
Iron Binding ◽  
Binding Ability ◽  
Amide Bonds ◽  
Plasma Half Life ◽  
Iron Chelator Deferoxamine

The traditional iron chelator deferoxamine (DFO) has been widely used in the treatment of iron overload disease. However, DFO has congenital disadvantages, including a very short circular time and non-negligible toxicity. Herein, we designed a novel multi-arm conjugate for prolonging DFO duration in vivo and reducing cytotoxicity. The star-like 8-arm-polyethylene glycol (8-arm-PEG) was used as the macromolecular scaffold, and DFO molecules were bound to the terminals of the PEG branches via amide bonds. The conjugates displayed comparable iron binding ability to the free DFO. Furthermore, these macromolecule conjugates could significantly reduce the cytotoxicity of the free DFO, and showed satisfactory iron clearance capability in the iron overloaded macrophage RAW 246.7. The plasma half-life of the 8-arm-PEG-DFO conjugate was about 190 times than that of DFO when applied to an intravenously administered rat model. In conclusion, research indicated that these star-like PEG-based conjugates could be promising candidates as long circulating, less toxic iron chelators.

Biological Systems to Assay Herbicidal Bleaching

1979 ◽  
Vol 34 (11) ◽  
pp. 1044-1046 ◽  
Author(s):  
Gerhard Sandmann ◽  
Karl-Josef Kunert ◽  
Peter Böger
Keyword(s):  
Graphical Method ◽  
Algal Cell ◽  
Biological Systems ◽  
High Sensitivity ◽  
Phycomyces Blakesleeanus ◽  
Dixon Plot ◽  
Physiological Conditions ◽  
Scenedesmus Acutus ◽  
Subsequent Application

Abstract Two strains of Scenedesmus acutus were found useful to study the influence of bleaching agents on either the greening process or the fully pigmented algal cell during growth. Both physiological conditions exhibit high sensitivity to bleaching herbicides. With this new assay, contrasting bleach­ing effects with the same compound can be found allowing differentiation of the herbicidal action of bleaching agents which apparently is a multifunctional one. Furthermore, the I50 can be determined rather rapidly in a simple graphical method by a Dixon plot. A subsequent application of bleaching herbicides to cultures of the fungus Phycomyces blakesleeanus rules out a possible specific action of the compounds assayed on chlorophyll or photosynthetic redox carriers. This latter assay can show whether or not the herbicides synthesis as is the case with difunon or SAN 9789.

Sign in / Sign up

Export Citation Format

Share Document