454 R. W. ESTABROOK 



intriguing to unravel the nature of the respiratory pigments present in various 

 sources, and indeed certainly makes the hypothesis of a unified cytochrome 

 complement untenable. At the present time the use of low temperature 

 spectroscopy appears to be the most promising approach to unravel the 

 complexity of variable pigment content in different types of materials. 



Trapped Steady States 



Recently Chance and Spencer (1959), and more recently Chance and 

 Bonner (unpublished), have succeeded in recording the difference spectrum 



T . 



AO.D.= 0.005 



I — I — \ — \ \ — I — I — \ — I 



500 550 



Fig. 19. The low temperature spectrum of the trapped steady states of rat 

 liver mitochondria. Samples of mitochondria, diluted in isotonic buffer, were 

 treated with substrate (state 4). One portion was treated with ADP to oxidize 

 the pigments to state 3. The samples were then rapidly cooled in liquid nitrogen 

 and the resulting difference in light absorption plotted as a function of wavelength. 



of pigments in various steady states of oxidation. This is shown in Fig. 19, 

 where the difference between the extent of cytochrome reduction in states 

 4 and 3 of phosphorylating rat liver mitochondria is presented. A clear 

 resolution of the crossover point between cytochromes b and c^ is demon- 

 strated. The combined increase in resolution of absorption bands coupled 

 with a means of rapidly trapping a steady state holds great promise in solving 

 in part the action of the respiratory pigments during oxidative phosphory- 

 lation. 



DISCUSSION 



The cooling of samples of reduced haemoproteins, in particular the cyto- 

 chromes, causes a sharpening of the associated absorption bands and a 

 splitting of some of these bands. The observed narrowing of the bands is 

 caused by restricting the energy loss from the excited vibrational states of 

 the absorbing molecule to the solvent molecules. The lowering of tempera- 

 ture therefore accomplishes in part a change comparable to the sharpening 

 of absorption bands seen when going from the liquid to the vapour state with 

 organic molecules such as benzene derivatives. 



The origin of the splitting of the absorption bands is the question of the 



