Cytocliromes Cooled in Liquid Nitrogen 457 



6. The samples of cytochrome c^ and c^ were prepared by Dr. R. Burris and N. Neumann 

 of the University of Wisconsin. 



7. Cytochrome Z?4 was prepared by Dr. Ryo Sato of Osaka University, Osaka, Japan. 



REFERENCES 



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Chance, B., Klingenberg, M. & Boeri, E. (1956). Fed. Proc. 15, 231. 



Chance, B. & Spencer, E. L. (1959). Disc. Faraday Soc. 27, 200. 



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Garfinkel, D. (1957). Arch. Biochem. Biophys. 71, 100. 



Keilin, D. (1925). Proc. roy. Soc. B. 98, 312. 



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Keilin, D. & Hartree, E. F. (1949). Nature, Loud. 164, 254. 



Keilin, D. & Hartree, E. F. (1955). Nature, Lond. 176, 200. 



Klingenberg, M. (1958). Arch. Biochem. Biophys. 75, 376. 



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TissiERES, A. (1956). Biochem. J. 64, 582. 



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DISCUSSION 

 'Trapped' Steady States 



By B. Chance (Philadelphia) 



Chance: Various experiments indicate that there is only a small change in the steady 

 state of cytochromes c, b and a in the transition from room temperature to liquid 

 nitrogen temperatures (77°K). Explanations for this are described elsewhere (Chance 

 and Spencer, Disc. Faraday Soc. 27, 200, 1959), and centre about the very similar 

 temperature coefficient of the inter-cytochrome reaction velocities. This method has 

 important applications in the identification of functional cytochromes and in locali- 

 zation of inhibitory interaction sites in the respiratory chain. 



Methods 



The method is described in Chance and Spencer (Disc. Faraday Soc. 27, 200, 1959), 

 and very briefly consists of modifications of Keilin's and Estabrook's procedures 

 suggested by Bonner and Yocum, namely the omission of glycerol. Modified cuvettes 

 for 1 and 3 mm paths are used. Instead of absolute spectra, difference spectra are 

 used. The steady-state oxidized reference material is washed and starved where 

 possible. It is strongly oxygenated to ensure an adequate supply of oxygen in the 



H 



