Carbon dioxide is vital to the chemistry of life processes including metabolism, cellular homoeostasis, and pathogenesis. CO
is generally unreactive but can combine with neutral amines to form ...carbamates on proteins under physiological conditions. The most widely known examples of this are CO
regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase and haemoglobin. However, the systematic identification of CO
-binding sites on proteins formed through carbamylation has not been possible due to the ready reversibility of carbamate formation. Here we demonstrate a methodology to identify protein carbamates using triethyloxonium tetrafluoroborate to covalently trap CO
, allowing for downstream proteomic analysis. This report describes the systematic identification of carbamates in a physiologically relevant environment. We demonstrate the identification of carbamylated proteins and the general principle that CO
can impact protein biochemistry through carbamate formation. The ability to identify protein carbamates will significantly advance our understanding of cellular CO
interactions.
The original version of this Article omitted the following from the Acknowledgements: 'This work was support by EPSRC grant EP/K504336/1 and Leverhulme Trust grant RPG-2016-017.' This has been ...corrected in both the PDF and HTML versions of the Article.
Carotenoids are a diverse family of naturally occurring pigments that are produced by plants, photosynthetic bacteria and certain fungi. Their biological roles within these organisms include the ...quenching of reactive oxygen species, acting as accessory pigments in photosystems and the colouration of flowers and fruits. Although mammals do not produce carotenoids, they may be obtained through the diet. Numerous health benefits in humans have been found to be associated with carotenoid containing diets, such as the maintenance of cardiovascular health and protection against degenerative eye diseases. These discoveries have led to an interest in ways to increase the stability of carotenoids within foodstuffs. Contrary to the vast majority of studies involving carotenoids, the detergency industry is interested in finding solutions to decolourise carotenoid containing stains. Carotenoid stains are usually composed of oily hydrophobic soils that adhere strongly to fabrics and sequester away from water. Due to the difficulty in complete removal of carotenoid stain material from fabric and the strong colouration of carotenoids, the detergency industry has adopted the approach of making the stain less perceivable through bleaching rather than attempting to eliminate all stain material. Although processes involved in the oxidative degradation of carotenoids are well established and there are numerous investigations of carotenoid decomposition in foodstuffs, quantitative studies of factors influencing the bleaching of carotenoids under conditions relevant to an aqueous washing environment are scarce. We have performed a quantitative study of the effect of fatty acid, surfactant and pH on the bleaching of β-carotene in oil-in-water emulsions. Experiments were monitored using UV-Vis spectrophotometry and focussed on quantifying initial rates of oxidation of fatty acid (a process closely related to carotenoid oxidative degradation) and the bleaching of β-carotene. Linoleic acid was found to autoxidise faster at lower pH while the bleaching of β-carotene in the presence of linoleic acid was found to proceed more faster at neutral pH. Experiments were performed in the absence and presence of lipoxygenases (LOXs) from soybean. These enzymes promote the bleaching of carotenoids by accelerating the formation of hydroperoxide bleaching species from polyunsaturated fatty acids. LOXs are known to be relatively slow, however, a study in the literature has reported that a bleaching synergy exists between two isozymes, LOX1 and LOX3. The activities of LOX1 and LOX3 linoleic acid oxidation and β-carotene degradation were quantified and found to be pH and surfactant dependent. Combinations of these enzymes were used to quantified this synergy under a range of conditions. To complement bleaching experiments in microemulsion solutions, work was performed at P&G Newcastle to investigate factors influencing the bleaching of carotenoid stains on fabrics. Stain decolourisation was quantified by stain removal index (SRI) values obtained by DigiEye. The effect of concentration of linoleic acid and α-tocopherol (a powerful antioxidant) in stain material was found to show correlation with results obtained from solution bleaching experiments and confirmed that the majority of fatty acid dependent bleaching of carotenoids on fabric is reliant on a radical mechanism. A study of the effect of fabric type and treatment discovered that stain removal in the wash was more facile on hydrophilic fabric, however, bleaching of carotenoid stain material after the wash appeared to be faster on hydrophobic materials. In experiments monitoring the bleaching of carotenoid containing microemulsions in the presence of fabric, cotton and polycotton were found to strongly inhibit the bleaching process while polyester slowed the rate but not the extent of bleaching. A side project, involved the preliminary study of the hydrolysis of triethyloxonium tetrafluoroborate, a commercially available alkylating agent, usually restricted to use in dry organic solvents. Preliminary results showed a pH independent region from pH 2 to 10. A pH dependent region was discovered above pH 10, however, reactions proceeded too rapidly to quantify rate constants. Hydrolysis rate constants were also measured in 50% acetonitrile with rate constants almost identical to those in 100% water.
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