476 APPENDIX 



the Chromatium cytochrome 553 become completely autoxidized during 

 purification and require no further treatment. Sodium dithionite is 

 used to reduce the cytochromes. For spectra of carbon monoxide com- 

 plexes, the reduced cytochrome solution is equilibrated with pure 

 carbon monoxide at 1 atmos. pressure in an anaerobic cuvette. The 

 cuvette contents are first degassed by evacuating with a vacuum line 

 and then flushing with high-purity argon. 



Dr. S. Taniguchi supplied /?. rubrum cytochromes and H. de Klerk 

 supplied the Rps. palustris cytochrome samples. 



RESULTS AND DISCUSSION 



Spectroscopic properties. 



Absorption maxima and corresponding molar extinction coefficients 

 of various states of cytochrome C2 from R. rubrum and Rps. palustris 

 plus the complex cytochrome 553 of Chromatium 2iVe listed in Table 1, 

 and corresponding data for cytochromoid C oi R. rubrum, Rps. 

 palustris and Chromatium are listed in Table 2, The spectra of these 

 cytochromes are presented in Figs, 1-6, respectively. These particu- 

 lar cytochromes were chosen because homogeneous samples were at 

 hand for preparation of the spectra. Few other pure cytochrome 

 preparations from photosynthetic bacteria have been reported; for 

 none of these were extinction coefficients determined, nor were dif- 

 ference spectra presented which might aid in the study of the cyto- 

 chrome in the native bound form. Incomplete spectra of /?/>s. spheroides 

 cytochrome 553 and cytochrome b are reproduced in Figs, 7 and 8, 

 respectively. In Table 3 are summarized some properties of incom- 

 pletely characterized purified cytochromes obtained from both purple 

 and green photosynthetic bacteria. 



Molar extinction values listed in Tables 1 and 2 are calculated in 

 two ways. For the R. rubrum and Chromatium proteins, values based 

 on dry weight and molecular weight determinations are presented. In 

 addition, values are calculated in terms of the heme concentration of 

 each cytochrome sample. Heme concentrations were estimated from 

 alkaline pyridine hemochromogen spectra, using Drabkin's value for 

 the molar extinction coefficient of the 550 m^ absorption peak of the 

 cytochrome c derivative, a^ x 10"^ liter/mol, cm, = 29 (9). To pre- 

 pare the pyridine hemochromogen derivatives, the samples were di- 

 luted in 0,1 M NaOH-25% v/v pyridine and then 1-2 mg, dithionite per 

 ml. was added. After a five-minute delay the absorption spectrum in 

 the visible region was measured vs. a solvent blank. In every instance 

 the a peak was located at 550 + 0,5 m/u, indicating that the proteins 

 contain heme groups like that of cytochrome c. The relatively close 

 agreement between values determined in these two ways supports the 

 notion that, if a rigorous determination of molecular weight is im- 



