Abstract
The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth rate of 0.1 h-1 are recorded. This is accomplished by transporting the emitted light from the spectrophotometer via glass fibers to one side of the chemostat vessel and detecting the transmitted light via a photomultiplier at the other side. The vessel itself is enclosed in a dark box, which contains mirrors on the inside surfaces. The reduction levels of cytochromes b and c during steady state in chemostat cultures are expressed as percentage absorbance of fully reduced cytochromes in the alpha-region of the spectrum. Steady state spectra are recorded in N2-fixing, succinate-limited continuous cultures of Azorhizobium caulinodans at dissolved oxygen tensions in the range between 0.1 and 3.5% O2. Spectra of fully reduced cytochromes are obtained on the basis of spectra recorded after having reached anoxic conditions by sparging pure nitrogen gas through the culture. These spectra of cytochromes b and c reduced by endogenous substrates are corrected as to give the spectrum of fully reduced cytochromes. The respective contributions of cytochromes b and c to spectra in the alpha-region are estimated by deconvolution using best-fit analysis. Using this in situ technique it is observed that at each dissolved oxygen tension the reduction level of the cytochromes b is higher than that of the cytochromes c.(ABSTRACT TRUNCATED AT 250 WORDS)
Original language | English |
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Pages (from-to) | 149-55 |
Number of pages | 7 |
Journal | Analytical Biochemistry |
Volume | 214 |
Issue number | 1 |
DOIs | |
Publication status | Published - Oct 1993 |
Keywords
- Cytochrome b Group
- Cytochrome c Group
- Nitrogen Fixation
- Oxidation-Reduction
- Oxygen
- Rhizobiaceae
- Spectrophotometry
- Journal Article
- Research Support, Non-U.S. Gov't