Multicomponent kinetic analysis and theoretical studies on the phenolic intermediates in the oxidation of eugenol and isoeugenol catalyzed by laccase.

Laccase catalyzes the oxidation of natural phenols and thereby is believed to initialize reactions in lignification and delignification. Numerous phenolic mediators have also been applied in laccase-mediator systems. However, reaction details after the primary O-H rupture of phenols remain obscure. In this work two types of isomeric phenols, EUG (eugenol) and ISO (trans-/cis-isoeugenol), were used as chemical probes to explore the enzymatic reaction pathways, with the combined methods of time-resolved UV-Vis absorption spectra, MCR-ALS, HPLC-MS, and quantum mechanical (QM) calculations. It has been found that the EUG-consuming rate is linear to its concentration, while the ISO not. Besides, an o-methoxy quinone methide intermediate, (E/Z)-4-allylidene-2-methoxycyclohexa-2,5-dienone, was evidenced in the case of EUG with the UV-Vis measurement, mass spectra and TD-DFT calculations; in contrast, an ISO-generating phenoxyl radical, a (E/Z)-2-methoxy-4-(prop-1-en-1-yl) phenoxyl radical, was identified in the case of ISO. Furthermore, QM calculations indicated that the EUG-generating phenoxyl radical (an O-centered radical) can easily transform into an allylic radical (a C-centered radical) by hydrogen atom transfer (HAT) with a calculated activation enthalpy of 5.3 kcal mol(-1) and then be fast oxidized to the observed eugenol quinone methide, rather than an O-radical alkene addition with barriers above 12.8 kcal mol(-1). In contrast, the ISO-generating phenoxyl radical directly undergoes a radical coupling (RC) process, with a barrier of 4.8 kcal mol(-1), while the HAT isomerization between O- and C-centered radicals has a higher reaction barrier of 8.0 kcal mol(-1). The electronic conjugation of the benzyl-type radical and the aromatic allylic radical leads to differentiation of the two pathways. These results imply that competitive reaction pathways exist for the nascent reactive intermediates generated in the laccase-catalyzed oxidation of natural phenols, which is important for understanding the lignin polymerization and may shed some light on the development of efficient laccase-mediator systems.

Fluorescent detection of lipid peroxidation derived protein adducts upon in-vitro cigarette smoke exposure.

Oxidative stress in biological systems can result in radical-induced lipid peroxidation (LPO), which can lead to the production of secondary reactive by-products such as 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), acrolein, and acetaldehyde. These deleterious compounds are known to react with and concomitantly modify nucleophilic amino acid residues on proteins. Oxidative stress induced by cigarette smoke (CS) has been put forth as a major mechanism for tobacco-induced pathologies. At present, there are few reliable biomarkers for measuring the extent of oxidatively-induced damage resulting from CS exposure in vivo. This study has utilized a previously reported CS exposure system to expose cultured cells in-vitro to whole CS and determine the extent of LPO resulting from CS exposure by quantifying the increase in HNE within the exposure media versus controls via gas chromatography mass spectrometry. Additionally, we obtained protein enriched cell lysate post-CS exposures and measured the fluorescent signal obtained via direct injection fluorescent analysis at 375 nm ex./415 nm em. This study determined that the fluorescent signal intensity was directly proportional to the quantity increase of HNE in CS exposed media. It further tested this correlation by performing HNE titration addition experiments to cultured cells and Western blot analysis on proteins obtained from cell lysates. Finally, the fluorescent signal increase from authentic BSA solutions incubated with increasing concentrations of HNE was measured. It is proposed that the fluorescent signal observed from the protein lysate of CS exposed cultured cells corresponds to the extent of biological damage resulting from secondary reactive by-products formed from LPO induced via CS exposure and represented by HNE. The fluorescent signals increased in intensity upon increasing CS dose up to 20 min and remained elevated over 24 h after cessation of CS exposure.