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Polyacrylamide usually is the material of choice for electrophoretic separation in slab gels, capillaries, and microfluidic devices. So far its polymerization requires anaerobic environments because oxygen impurities inhibit or even terminate the polymerization reaction of acrylamide. Here, it is demonstrated that gel precursor solutions with glycerol contents above 20 vol% enable direct atmospheric photopolymerization of acrylamide with no need for sealing or degassing the solution in advance. The positive effect of glycerol on the polymerization reaction is proven by simulation-validated electron paramagnetic resonance measurements. Nuclear magnetic resonance reveals that glycerol does not interfere with the reaction indicating that the observed enhancement in polymerization is owed to the low oxygen solubility of aqueous glycerol mixtures. Glycerol concentrations of >60 vol% in the gel precursor solution enable complete polymerization of volumes down to 5 nL within less than 5 s. This enables using liquid handling robots to fabricate channel-free open microfluidic structures of solid polyacrylamide hydrogel in a low-cost automated manner in a standard lab environment. [ABSTRACT FROM AUTHOR]

This work characterizes the properties of deposited plasma polymer that has been generated using three different low-pressure, low-temperature plasma modes: magnetron-enhanced audio frequency (15 kHz), magnetron-enhanced radio frequency (13.56 MHz), and radio frequency (13.56 MHz). A correlation between the applied plasma modes, the concentration of generated radicals in the deposited nanofilms, and the radical decay kinetics was found and is discussed. In addition, the role of the plasma driving frequency with respect to the properties of the films, in particular their density and their barrier properties, has been elucidated. [ABSTRACT FROM AUTHOR]

Exploring enzymatic mechanisms at a molecular level is one of the major challenges in modern biophysics. Based on enzyme structure data, as obtained by X-ray crystallography or NMR spectroscopy, one can suggest how substrates and products bind for catalysis. However, from the 3D structure alone it is very rarely possible to identify how intermediates are formed and how they are interconverted. Molecular spectroscopy can provide such information and thus supplement our knowledge on the specific enzymatic reaction under consideration. In the case of enzymatic processes in which paramagnetic molecules play a role, EPR and related methods such as electron-nuclear double resonance (ENDOR) are powerful techniques to unravel important details, e.g. the electronic structure or the protonation state of the intermediate(s) carrying (the) unpaired electron spin(s). Here, we review recent EPR/ENDOR studies of blue-light active flavoproteins with emphasis on photolyases that catalyze the enzymatic repair of UV damaged DNA, and on cryptochrome blue-light photoreceptors that act in several species as central components of the circadian clock. [ABSTRACT FROM AUTHOR]

(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH, which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH, namely those of H8α and H1', yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His354 and His358, in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His358 is deprotonated, whereas His354 is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct. [ABSTRACT FROM AUTHOR]

Electron transfer reactions play vital roles in many biological processes. Very often the transfer of charge(s) proceeds stepwise over large distances involving several amino acid residues. By using time-resolved electron paramagnetic resonance and optical spectroscopy, we have studied the mechanism of light-induced reduction of the FAD cofactor of cryptochrome/photolyase family proteins. In this study, we demonstrate that electron abstraction from a nearby amino acid by the excited FAD triggers further electron transfer steps even if the conserved chain of three tryptophans, known to be an effective electron transfer pathway in these proteins, is blocked. Furthermore, we were able to characterize this secondary electron transfer pathway and identify the amino acid partner of the resulting flavin-amino acid radical pair as a tyrosine located at the protein surface. This alternative electron transfer pathway could explain why interrupting the conserved tryptophan triad does not necessarily alter photoreactions of cryptochromes in vivo. Taken together, our results demonstrate that light-induced electron transfer is a robust property of cryptochromes and more intricate than commonly anticipated. [ABSTRACT FROM AUTHOR]