458 Discussion 



filling and freezing interval. The steady-state reduced material is similarly oxygenated 

 and is treated with substrate just prior to pipetting into the cuvette. The cuvette may 

 be pre-chilled so that freezing occurs very nearly on contact with the cooled surfaces. 

 The difference spectrum is then plotted in the split-beam recorder (see Chance, Methods 

 in Eiizymol. 4, 273, 1957). 



Identification of Respiratory Enzymes 



It was pointed out in the discussion of Chaix's paper that the distinction between 

 respiratory and accessory pigments (for definition see Chance, this volume, p. 476), 

 could be simply made by observation of their steady-state reduction. Trapping of 

 this steady state at liquid nitrogen temperatures has the great advantage of com- 

 bining the precise identification of cytochrome type afforded by the sharpening and 

 splitting of the bands at low temperatures with the test for respiratory function. An 

 example of this technique is aff"orded by Fig. 3 of Chance (this volume, p. 607) which 

 illustrates a frozen steady state for succinate-treated ox heart mitochondria. In 

 addition to the reduction of cytochromes a and c to values characteristic of active 

 electron transfer, we find the steady-state reduction of cytochrome c^ to be readily 

 observed for the first time in this material and, in addition, can calculate its extent of 

 reduction as a percentage of the fully reduced material. This value is very close to 

 that of cytochrome c. 



Inhibitory Interaction Sites 



The cross-over theorem (see Chance, this volume, p. 607) affords a unique method 

 for the localization of inhibitory interaction sites in the respiratory chain. The changes 

 in steady state of cytochromes b and c may be small and difficult to m.casure, and in 

 any case, cytochrome q cannot be discerned at room temperatures. 



Tiie application of trapped steady states to the study of cross-over behaviour is 

 illustrated by several figures given in the oral presentation, one of which is included 

 in the text of this volume (Estabrook (Fig. 19), this volume, p. 454). We can demon- 

 strate the state 2-A and 3^ transitions in the presence and absence of azide. The 

 participation of cytochrome c^ in these transitions is clearly shown and a cytochrome 

 b-Ci crossover is demonstrated (see Chance, this volume, p. 607). 



The technique has also been applied to cross-over phenomena in intact cells and the 

 increased reduction of cytochrome b and c characteristic of the inhibited phase of 

 glucose metabolism can readily be observed, although these changes are too small to 

 allow a resolution of cytochrome q. 



One of the greatest limitations of the low-temperature technique, namely that it 

 affords only identification and gives no indication of function, appears to be overcome 

 with the trapped steady-state method. It is probable that most of the functional and 

 identification test for cytochromes will be done at low temperatures in the future. 



Low Temperature Absorption Spectra of Cytochromes in Relation to Structure 

 Possible Differences in the Prosthetic Groups ofc-type Cytochromes 

 Morton: With reference to the differences in the low temperature absorption spectra 

 noted by Estabrook, it seems possible that there may be small differences in the 

 prosthetic groups of cytochrome c from various sources. Not only does yeast cyto- 

 chrome c differ from heart-muscle cytochrome c in absorption spectra both at room 

 temperature and at — 190°C, but the prosthetic group of native yeast cytochrome c 

 is more readily split by treatment with silver sulphate in acetic acid, as previously 

 noted (Armstrong, Coates and Morton, this publication, p. 386). In one sample of 

 aggregated yeast cytochrome c, Armstrong in my laboratory observed that the 

 prosthetic group was split by treatment of the cytochrome with 2 n acetic acid only. 

 Unfortunately, treatment with acetone-HCl was not investigated. Falk, Appleby and 

 Porra {Soc. exp. Biol. Sympos. 13, 73, 1959) have reported the isolation from effective 



