578 E. C. Slater and J. P. Colpa-Boonstra 



shorter wavelength when haemoglobin was added. Under the conditions of 

 these measurements, cytochrome a^ does not interfere, since it remains in the 

 oxidized state and does not combine with CO. 



The amount of oxymyoglobin present was 0-2-0-3 /^mole/g protein (cf. 

 cytochromes c + Cj + protohaematin compounds, about 1-5/imoles/g 

 protein). 



Oxymyoglobin interferes in measurements of the difference spectra (re- 

 duced minus oxidized) when succinate is substrate, since it is deoxygenated 

 to myoglobin when the suspension goes anaerobic. This can be eliminated 

 by previous treatment of the preparation with nitrite to oxidize the oxymyo- 

 globin to metmyoglobin, as suggested by Chance (1952). Metmyoglobin is 

 not reduced by succinate in the presence of the heart-muscle preparation. 



Other Cytochromes 



Although the absorption bands of the cytochromes are sharp, they are 

 sufficiently close together to cause considerable overlapping. It is desirable, 

 therefore, to choose a wavelength where changes of absorbancy are due only 

 to cytochrome b, and not to the other cytochromes. Holton (1955) has 

 introduced a method, which enables this to be determined. In heart-muscle 

 preparation oxidizing succinate in the presence of air, the cytochromes are 

 largely oxidized, and become reduced when the suspension becomes anaerobic 

 (Chance, 1952). Holton (1955) found that, in the presence of malonate, the 

 reduction of cytochrome b so lagged behind that of the other cytochromes 

 that in the region of the peaks of this cytochrome the first change was a 

 decrease of absorbancy caused by reduction of the other cytochromes, 

 followed by an increase of absorbancy caused by reduction of cytochrome b. 

 By carrying out the measurements at different wavelengths, it was possible 

 accurately to determine the wavelength at which the first decrease of ab- 

 sorbancy did not occur. This represents the isosbestic points of all the other 

 cytochromes taken together. These were found to be at 434 m/j, and 558 nyi 

 (Colpa-Boonstra and Holton, 1959). 



Chance (1958) introduced another method of ehminating the contribution 

 of the other cytochromes to the spectrum. This makes use of the fact that, 

 in the presence of cyanide, ascorbate reduces all the cytochromes except b. 

 This method is very suitable for accurately determining the spectrum ob- 

 tained by addition of succinate, and by the subsequent addition of antimycin. 

 Neither spectrum obtained is influenced by the presence of oxymyoglobin or 

 metmyoglobin, because the latter is not reduced and the former remains 

 oxygenated since oxygen is not consumed by the solution in the presence of 

 cyanide. However, we do not agree with Chance that the spectrum obtained 

 by subsequent addition of NagSgO^ is not affected by the presence of these 

 compounds; indeed, both would be reduced to myoglobin. This would be 

 expected to lead to increased absorption in the regions 435 mfi and 556-566 



