Quantitating carbon monoxide production from heme by vascular plant preparations in vitro
ABSTRACT Heme in animals is mainly degraded enzymatically, producing a predictable amount of carbon monoxide (CO). Under some conditions, alternative sources of CO production are important, such as lipid peroxidation and photo-oxidation. Less is known about CO production in plants as a reflection of enzymatic activity or coupled oxidation, but a sensitive assay for CO production in plants would be a valuable tool to explore the various sources in plants as the conditions of the reactions and mechanisms are defined. Using gas chromatography, we determined the requirements for heme-supported in vitro CO generation by exogenous reactants (NADPH, tissue supernatant, oxygen), optimum reaction conditions (time, temperature, pH, light), and effects of various cofactors and substrates using supernatants from Spinacia oleracea (spinach) leaf and Solanum tuberosa (potato) tuber homogenates. We then determined the CO production rate distribution between organ (root, stem, leaf, flower, fruit) supernatants in a number of commercially available plant species. CO production ranged from 4-65 nmol CO/h/g fresh weight and occurred in all vascular plant tissues examined, with the highest rates in chloroplast-containing tissues. In spinach leaves, CO production was concentrated (>2-fold) in the particulate fraction, whereas in potato tubers, the particulate fraction accounted for <50% of the rates in homogenates. We conclude that gas chromatography is uniquely suited for the determination of CO production in pigmented, heterogeneous plant tissue preparations.
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ABSTRACT: Carbon monoxide is an endogenous small-signalling molecule in the human body, produced by the action of heme oxygenase (HO) on heme. Since it is very difficult to apply safely as a gas, solid storage and delivery forms for CO are now explored. Most of these CO-releasing molecules (CORMs) are based on the inactivation of the carbon monoxide by coordination to a transition metal centre in a prodrug approach. After a brief look at the potential cellular target structures of carbon monoxide, an overview of the design principles and activation mechanisms for CO release from a metal coordination sphere is given. Endogenous and exogenous triggers discussed include ligand exchange reactions with medium, enzymatically induced CO-release (ET-CORMs), and photoactivated liberation of carbon monoxide (PhotoCORMs). Furthermore, the attachment of CORMs to hard and soft nanomaterials to confer additional target specificity to such systems is critically assessed. A comparison of analytical methods for the study of CO release stoichiometry and kinetics as well as the tracking of carbon monoxide in living systems by fluorescent probes concludes this review. CO-releasing molecules are very valuable tools to study carbon monoxide bioactivity and might lead to new drug candidates, but in the design of future generations of CORMs, particular attention has to be paid to their drug-likeness and the tuning of the peripheral "drug sphere" for specific biomedical applications. Further progress in the field will thus critically depend on a close interaction of synthetic chemists with reseachers exploring the physiological effects and therapeutic applications of carbon monoxide.British Journal of Pharmacology 03/2014; 172(6). DOI:10.1111/bph.12688 · 5.07 Impact Factor
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ABSTRACT: The exogenous application of carbon monoxide (CO) is a valuable strategy which enables study of the effects under different stress conditions. However, in this experimental model a true endogenous CO production by plants can not be measured. In this work, so as to achieve an elevated sensitivity and to avoid invasive techniques, we quantify the endogenous CO production by tissues in salt-treated soybean plants through gas chromatography coupled to a reduction gas detector. This technique allows short and room temperature incubation of intact tissues and homogenates. We found that a 200 mM NaCl treatment induces total CO production in leaves and roots. The sensitivity of the technique offers no correlation between this increment and heme oxygenase (HO) activity measured as a function of CO production. We also found that untreated soybean plants continue to produce significant CO levels up to 7 days post planting, after which CO content decreases to a third and remains constant in the next days. However, HO activity does not change throughout these days. The data here reported shows that HO activity is not the main source of CO in soybean plants. We discuss alternative sources that could be implicated in this production. Taking our own results and data reported by other colleagues, we propose lipid peroxidation and ureide metabolism as potential sources of CO.Environmental and Experimental Botany 06/2014; 102. DOI:10.1016/j.envexpbot.2014.01.012 · 3.00 Impact Factor