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The cold atmospheric pressure plasma-generated species superoxide, singlet oxygen and atomic oxygen activate the molecular chaperone Hsp33

Cold atmospheric pressure plasmas are used for surface decontamination or disinfection, e.g. in clinical settings. Protein aggregation has been shown to significantly contribute to the antibacterial mechanisms of plasma. To investigate the potential role of the redox-activated zinc-binding chaperone Hsp33 in preventing protein aggregation and thus mediating plasma resistance, we compared the plasma sensitivity of wild-type E. coli to that of an hslO deletion mutant lacking Hsp33 as well as an over-producing strain. Over-production of Hsp33 increased plasma survival rates above wild-type levels. Hsp33 was previously shown to be activated by plasma in vitro. For the PlasmaDerm source applied in dermatology, reversible activation of Hsp33 was confirmed. Thiol oxidation and Hsp33 unfolding, both crucial for Hsp33 activation, occurred during plasma treatment. After prolonged plasma exposure, however, unspecific protein oxidation was detected, the ability of Hsp33 to bind zinc ions was decreased without direct modifications of the zinc-binding motif, and the protein was inactivated. To identify chemical species of potential relevance for plasma-induced Hsp33 activation, reactive oxygen species were tested for their ability to activate Hsp33 in vitro. Superoxide, singlet oxygen and potentially atomic oxygen activate Hsp33, while no evidence was found for activation by ozone, peroxynitrite or hydroxyl radicals.

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Plasma Source Name
Plasma Source Application
Plasma Source Specification
Plasma Source Properties
DBD: V(RMS) = 13.5 kV, trigger frequency = 300 Hz, diameter = 20 mm µAPPJ: frequency = 13.56 MHz, V(RMS) = 230 V
English (United States)
Plasma Source Procedure
If not indicated otherwise, plasma treatment was carried out using the PlasmaDerm dielectric barrier discharge (DBD) with an electrode diameter of 20 mm. Plasma was ignited in ambient air using VRMS=13.5 kV and a trigger frequency of 300 Hz. Samples (40 μl) were placed on top of a metal plate on a grounded counter electrode with a 1 mm distance to the driven electrode. In addition, a microscale atmospheric pressure plasma jet (μAPPJ) was used. Helium (1.4 slm, 5.0 purity) with varying oxygen admixtures (4.8 purity) was used as feed gas. Plasma ignition was driven with 13.56 MHz and 230 VRMS. The distance of the sample to the nozzle of the jet was varied from 0 to 20 mm.
Plasma Medium Name
Plasma Medium Properties
Plasma Target Name
Contact Name
Bandow, Julia E.
Plasma Target Properties
Heat shock protein 33 (Hsp33), purified from Escherichia coli expression strain in 40 mM KPi buffer, pH 7.5
Plasma Target Procedure
Prior to any experiment employing Hsp33, the protein was reduced (Hsp33red) by adding 5 mM DTT and 115 μM ZnCl2 to 5 mg ml−1 Hsp33. This was followed by incubation at 37°C for 2 h. Chemically oxidized Hsp33 (Hsp33HOCl) was used as a positive control. To generate it, sodium hypochlorite was diluted in water yielding an equimolar mixture of hypochlorous acid (HOCl) and its conjugate base (OCl−). Hsp33red was oxidized by incubation with a 50-fold molar excess of HOCl at 30°C and 300 rpm for 10 min. After reduction or oxidation, Hsp33red and Hsp33HOCl were purified using the Micro Bio-SpinColumns-P30 according to manufacturers’ instructions.
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Project Contact Name: 
Bandow, Julia E.