Anaerobic samples

FIGURE 3.8 Outline of the vacuum/gas manifold for anaerobic sample preparation. 

Check whether room-temperature sample loading is required (this can destroy your anaerobic sample). 

However, near-stoichiometric Fe " ion binding to NifU-1 or NifU was observable only in experiments conducted at 2°C in anaerobic samples that had been pretreated with dithiothreitol to ensure reduction of any intrasubunit or intersubunit disulfides. At room temperature, <10% of the NifU-1 or NifU was in a Fe bound form, and colorimetric analysis indicates that the remainder of the Fe is in solution was in the form of free Fe " ion. Hence this mononuclear Fe -bound species is more likely to be an intermediate in the reduction of Fe ion by NifU or NifU-1 rather than an initial step in cluster assembly on the NifU-1 domain of NifU. In this connection, it is important to note that Fe is rapidly reduced to Fe by cysteine in aqueous solution (Schubert, 1932). The physiological significance (if any) of the apparent ferric reductase activity associated with the NifU-1 domain of NifU remains to be established. 

All Ni(OEP) and Ni(PP) samples (obtained from Porphyrin Products) were prepared (0.2-0.5 mM) in neat, spectral grade solvent (used without further purification) and were deoxygenated by purging with oxygen-free N2 S s. No differences in the spectra were noted for aerobic and anaerobic samples. 

Anaerobic samples were prepared by degassing the solvent by three freeze-thaw cycles while solids and NMR tubes were degassed by placing under vacuum, then storing in a nitrogen atmosphere. If oxygen is not carefully excluded, the ferrous porphyrin obtained by the autoreduction may be rapidly oxidized to the oxo-bridged dimer. 

Ba92 S. Bandow et ai, Anaerobic Sampling and Characterization of Lanthanofullerenes Extraction of LaCye and other LaC2n> J- Phys. Chem. 96, 9609-9612 (1992). 

Figure 9-3B shows a Mossbauer spectrum, recorded in the presence of a weak applied magnetic field, of a sample of E. coli whole cells, anaerobically grown, in which FNR has been over-expressed (FNR+). For comparison. Figure 9-3A shows again the quadrupole doublet of the isolated protein, discussed in Section 9.1, shown here to emphasize that this feature represents the dominant contribution in the spectrum of the anaerobic whole cells containing over-expressed FNR. As indicated by the dotted vertical lines, the position of the central doublet in Figure 9-3B coincides with that of the doublet in Figure 9-3A. Analysis of the spectrum of Figure 9-3B suggests that the [4Fe-4S] + cluster of the FNR transcription factor represents about 20% of the total iron in this anaerobic sample.

Photosystem 2 reaction centre complexes were prepared from pea chloroplasts as previously described [5,7], Samples were suspended in a buffer of 50 mM Tris-CI (pH8 at room temperature) containing 2 mM dodecylmaltoside to give a final chlorophyll concentration of 10 pgml (unless otherwise stated). Anaerobic conditions were achieved as described in [5,7]. All experiments were performed on anaerobic samples at a temperature of 277 K unless otherwise stated. 

Anaerobic Environments. When handling anaerobic samples, extreme care is necessary to avoid contact with oxygen during sampling, transport, storage, and elution (Wallmann et al. 1993). Another method is to simulate the transition from oxic to anoxic environments by elution tests. Although the observed effects may be significant (e.g. release of As and Fe by reduction of Fe(III) to Fe(II) Cu and Cd fixation by sulfide formation), the time necessary for redox experiments is in the order of weeks, as compared to hours typical for pH experiments. 

When 5-enolpyruvylshikimate-3-phosphate was added to an anaerobic sample of reduced FMN and chorismate synthase, the substrate was immediately converted to chorismate, followed by slow formation of a flavin radical. Similarly, when (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate was added to an identical sample, rapid formation of the radical was observed. It was suggested that the reduced flavin/enzyme complex is not stable in the presence of substrate or substrate analogue and is oxidized by an unidentified species to yield the flavin radical. 

Control experiments (A) aerobic and (B) anaerobic samples. Addition of TNBT ( 16 p M) under anaerobic conditions (C) anaerobic sample. 

Addition of TNBT under aerobic conditions (D) aerobic and (E) anaerobic samples. 

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