17 research outputs found
Fingerprinting antioxidative activities in plants
<p>Abstract</p> <p>Background</p> <p>A plethora of concurrent cellular activities is mobilised in the adaptation of plants to adverse environmental conditions. This response can be quantified by physiological experiments or metabolic profiling. The intention of this work is to reduce the number of metabolic processes studied to a minimum of relevant parameters with a maximum yield of information. Therefore, we inspected 'summary parameters' characteristic for whole classes of antioxidative metabolites and key enzymes.</p> <p>Results</p> <p>Three bioluminescence assays are presented. A horseradish peroxidase-based total antioxidative capacity (TAC) assay is used to probe low molecular weight antioxidants. Peroxidases are quantified by their luminol converting activity (LUPO). Finally, we quantify high molecular weight superoxide anion scavenging activity (SOSA) using coelenterazine.</p> <p>Experiments with <it>Lepidium sativum </it>L. show how salt, drought, cold, and heat influence the antioxidative system represented here by TAC, LUPO, SOSA, catalase, and glutathione reductase (GR). LUPO and SOSA run anti-parallel under all investigated stress conditions suggesting shifts in antioxidative functions rather than formation of antioxidative power. TAC runs in parallel with GR. This indicates that a majority of low molecular weight antioxidants in plants is represented by glutathione.</p> <p>Conclusion</p> <p>The set of assays presented here is capable of characterising antioxidative activities in plants. It is inexpensive, quick and reproducible and delivers quantitative data. 'Summary parameters' like TAC, LUPO, and SOSA are quantitative traits which may be promising for implementation in high-throughput screening for robustness of novel mutants, transgenics, or breeds.</p
A novel fluorescent pH probe for expression in plants
BACKGROUND: The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. RESULTS: pHluorins have a higher pH-sensitivity. However, Pt-GFP has a broader pH-responsiveness, an excellent dynamic ratio range and a better acid stability. We demonstrate how Pt-GFP expressing Arabidopsis thaliana report cytosolic pH-clamp and changes of cytosolic pH in the response to anoxia and salt-stress. CONCLUSION: Pt-GFP appears to be the better choice when used for in vivo-recording of cellular pH in plants
Peroxide-Induced Liberation of Iron from Heme Switches Catalysis during Luminol Reaction and Causes Loss of Light and Heterodyning of Luminescence Kinetics
The
peroxidation of luminol yields bright luminescence when the
reaction is catalyzed by heme proteins. However, an excess of peroxide
leads to less light and altered luminescence kinetics, an effect commonly
referred to as “suicide inactivation”. The aim of this
study is to present the molecular processes causing this effect. A
comprehensive set of data reported here demonstrates that suicide
inactivation is due to a peroxide-induced liberation of iron from
its coordinating porphyrin. Liberated iron launches catalysis of the
reaction at much lower efficiency. The light-yielding efficiencies
of different organic and inorganic catalysts are precisely quantified
and compared. It is shown that the catalysis by free iron involves
superoxide. This is explained by the formation of a ferryl-oxo-iron
complex. In this context, a complete reaction mechanism involving
a modified Fenton–Haber–Weiss cycle is proposed for
the first time. The switch from the highly efficient biogenically
catalyzed luminescence to a less efficient inorganically catalyzed
reaction is accompanied by a transition from “flash-type”
to “glow-type” luminescence kinetics. Ethylenediaminetetraacetic
acid-mediated chelation of iron is used to demonstrate this effect
and to separate both kinetics. The explanation of kinetic heterodyning
is underpinned by mathematical modeling. The results are able to explain
the as yet unexplained phenomena discussed in the less recent literature
and to settle disputes about them. It is concluded that peroxide concentrations
exceeding the level tolerated by the catalyzing heme protein negatively
impact performance and precision of luminol-based assays, where the
light yield is used as a quantitative measure for analyte concentrations
Redox Modulators Determine Luminol Luminescence Generated by Porphyrin-Coordinated Iron and May Repress “Suicide Inactivation”
Iron porphyrin catalysts
of the luminol reaction (horseradish peroxidase,
hemoglobin, cytochrome c, and hemin) interact with
diverse reducing compounds. Here, it is demonstrated how the chemiluminescence
yield is modulated by such interactions. The compounds accepted as
substrates protect the catalyst against the “suicide inactivation”
caused by high peroxide concentrations. The reducing agents not accepted
by the catalyst inhibit light production either by generating a futile
redox cycle of the luminophore or by irreversibly inactivating the
catalytic center. In the case of a futile cycle, light emission resumes
as soon as the reducing agents in the reaction are consumed, whereas
with an irreversible inactivation, light emission does not recover.
The characteristics of luminescence enhancement and quenching depending
on interfering agents are also reported here. They reveal details
about the relative redox potentials of the involved compounds. It
is discussed how this should be considered when the luminol reaction
is used for quantitative analyses and when unpurified samples with
a broad compound matrix are to be assayed