Fluorescence of Naphthol AS-MX is Readily Detectable in Dioxane Mixtures

Numerous enzymes have been demonstrated to be active in non-aqueous solutions, yet the utility of phosphatases under such conditions has been difficult to determine. Here, we demonstrate the ability to fluorescently detect naphthol AS‑MX in high percentages 1,4-dioxane with a fluorescence differential compared with naphthol AS‑MX phosphate. While intensities and maximum fluorescence wavelengths changed depending on solvent conditions, these results demonstrate this system’s potential for testing phosphatase activity in high amounts of dioxane

Wheat Germ Acid Phosphatase Activity in High Percentages of Organic Solvents

While enzymes canonically operate in aqueous environments, several have been shown to also function in non-aqueous solvents and/or solvent mixes. Phosphatases, for example, have been previously shown to hydrolyze phosphates in mixtures containing modest amounts of certain organic solvents. Here, detecting the fluorescent dephosphorylation product naphthol AS-MX with fluorescence spectroscopy (ex: 388 nm, em: 512 nm) has shown commercial wheat germ acid phosphatase to be active in high amounts (up to ~70% by volume) of 1,4-dioxane, 1,2- dimethoxyethane, 2-methoxyethanol, dimethyl sulfoxide, and acetonitrile when the aqueous component was comprised of Tris buffer (pH 7). These solvent mixtures were also physically characterized to better understand differences in both relative naphthol AS-MX fluorescence and wheat germ acid phosphatase activity across the different solvent mixtures investigated. These results suggest that phosphatase activity can be detected with less water that previously published

5,7,3′,4′-Hydroxy substituted flavonoids reduce the heme of cytochrome c with a range of rate constants

Flavonoids are antioxidants known to be abundant in edible plants. Seven 5,7,3′,4′-tetrahydroxy substituted flavonoids representing each major flavonoid class were used as cytochrome c reductants to systematically investigate the redox role of their C-rings. Additional examples of flavonoids and benzenediols were investigated to better understand the role of the B-ring. Pseudo-first order rate constants (k obs )and apparent bimolecular rate constants (k app )values were calculated from spectroscopic measurements. Of the seven flavonoids tested, five yielded measurable observed reduction rate constants. Butein (a chalcone)had the highest apparent bimolecular rate constant (k app ), followed by taxifolin (a flavanonol), catechin (a flavanol), eriodictyol (a flavanone), and luteolin (a flavone). Quercetin (a flavonol)and cyanidin (an anthocyanidin), however, reduced cytochrome c but k app rate constants were unable to be calculated. Neither this trend nor trends in observed rate constants correlated with flavonoid pK a , solvent accessible surface area, polar surface area, reduction potential, antioxidant ability, resonance, or radical scavenging efficiency. Weak correlation, however, was found with degrees of freedom and the number of redox involved electrons. While some cytochrome c reduction rates have been reported, this study is the first to systematically investigate the role of the structure of the flavonoid C-ring across a full set of flavonoids with identical B-rings