Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24-and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/ AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology.A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H 2 O 2 within the catalase-and peroxidase-deficient periplasmic space of E. coli.

To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases. p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide. Furthermore, p20 exerted the most potent lipid peroxidase activity. Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress. Expression level of p20 during anaerobic growth was sustained at the ϳ50% level compared with that of the aerobic growth. Viability of aerobic p20⌬ without glucose was reduced to the ϳ65% level of isogenic strains, whereas viability of aerobic p20⌬ with 0.5% glucose supplement was sustained. The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth. At the stationary phase, the viability of p20⌬ was down to ϳ10% level of parent strains. An analysis of the protein carbonyl contents of p20⌬ as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20⌬ was caused by cumulative oxidative stress. P20⌬ showed hypersensitivity toward membrane-soluble organic hydroperoxides. An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20⌬ demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane. Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats.

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.

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