474 



Discussion 



cytochrome b^ (557 m//) changes its oxidation-reduction level in synchronism with the 

 cytochromes of the respiratory chain. 



Other, much earlier work clearly shows what we now term the cytochrome 65 

 component to be reduced in liver cells. MacMunn (Phil. Trans. Roy. Soc. London 177, 

 267, 1886) and Keilin and Hartree (Proc. roy. Soc. B 129, 277, 1940) record reduced 

 bands of this component in liver. Thus, cytochrome b„ is accessible to reducing 

 substances, presumably cytoplasmic reduced diphosphopyridine nucleotide (DPNH), 

 but not to the mitochondrial oxidase, in accord with studies of isolated mitochondria 

 (Chance and Williams, /. biol. Chem. 217, 395, 1955). 



Isolated liver cells — If, instead of the tissue, we use a liver cell suspension (work in 

 collaboration with W. D. Rutter), we find in the low temperature 'absolute' spectra 

 of Fig. 2, the absorption bands of reduced cytochromes c, q, b and o + Og (dashed 



Normal Liver Cells 

 Absolute" Low temperature 

 Spectra 



500 550 600 



Fig. 2. 'Absolute' low temperature spectra of succinate-reduced (dashed 

 trace) and dithionite-reduced (solid trace) liver cell suspensions. (Expt. 885a). 



trace) but no b^ band. If dithionite is added (solid trace), the absorption of the b 

 components increases considerably and the bands of cytochrome b^ can be identified. 

 Thus it appears that some if not all of the 65 component is oxidized in the liver cell 

 suspension and can be reduced only in the presence of dithionite, in contrast with the 

 results on solid tissues. It is possible that cytoplasmic reducing substances, presumably 

 DPNH, have been oxidized or removed in the preparation procedure. 



A further examination of the cytoplasmic pigments of a liver cell suspension at low 

 temperatures is aff"orded by Fig. 3 in which the oxidized minus succinate-reduced 

 spectrum of the respiratory pigments (solid trace) is compared to that corresponding 

 to the difference between succinate-reduced and DPNH-reduced cells (dashed trace). 

 In the latter, the spectrum of the b components appears, showing the a^-band of 

 cytochrome b-^. (The slight difference in the maxima of Figs. 2 and 3 can be attributed 

 to differences between an absolute (Fig. 2) and a difference (Fig. 3) spectrum.) 



The room-temperature difference spectrum for the same conditions (Fig. 4) does 

 not show the bands described by Williams and myself (Chance and Williams, /. biol. 

 Chem. Ill, 395, 1955) at 426 and 557 mji, but instead shows an apparently different 

 'cytoplasmic pigment' with absorption bands at 423-5 and 555 m/t. The shoulder on 

 the long wavelength side of the oc-band suggests the presence of some cytochrome 65. 



Microspectrophotometry — To localize the pigments in question more accurately, 

 we have constructed a microspcctrophotometer capable of recording spectra of 

 respiratory and accessory pigments in situ (Chance, Perry, Akerman and Theorell, 

 Rev. Sci. Instrum. 30, 735, 1959). Figure 5 shows a spectrum of an area 1-5 /t in dia- 

 meter in a single cell teased from a portion of liver tissue after centrifugation of the 

 tissue and rendered anaerobic due to its own respiration (Theorell and Chance, Exptl. 

 Cell Research 20,43, 1960). Traces 1 represent 'absolute' spectra of various portions 

 of the cell containing the mitochondrial granules; traces 2 represent portions of the 



