SPECTROPHOTOMETRIC STUDIES OF 

 CYTOCHROMES COOLED IN LIQUID NITROGEN 



By R. W. ESTABROOK 



Johnson Foundation for Medical Physics, 

 University of Pennsylvania, Philadelphia 4 



INTRODUCTION 

 One of the primary problems associated with the studies of biological 

 oxidations is the recognition and identification of those components partici- 

 pating in the electron transport chain during the oxidation of substrates by 

 oxygen. The classical spectroscopic studies of Keilin (1925) showed the 

 presence of absorption bands characteristic for specific intracellular haemo- 

 proteins, which he called cytochromes. These studies, coupled with the 

 considerable numbers of enzymic tests carried out by Keilin and his collabora- 

 tors, have served as the foundation for our present understanding of cellular 

 respiration. The fact that the various haemoproteins have, in their reduced 

 form, specific absorption bands in the visible spectra has served as the means 

 of identification, thus permitting an investigation into the mode of interaction 

 of cytochromes. An excellent summary of the spectral properties of cyto- 

 chromes has recently been published by Morton (1958). 



One of the more recent developments in the spectroscopic identification of 

 the cytochromes are the observations, first pubhshed by Keilin and Hartree 

 in 1949, describing the remarkable effect of lowering the temperature on the 

 sharpening of the associated absorption bands of reduced cytochromes — thus 

 permitting the resolution of overlapping absorption bands. This effect 

 coupled with an intensification resulting from the devitrification of the 

 glycerol media employed by Keilin and Hartree (1949) permitted them to 

 recognize and characterize cytochrome c^ (Keilin and Hartree, 1955) having 

 in vitro properties which suggest its function as a member of the respiratory 

 chain, thus confirming the earlier contention of Yakushiji and Okunuki (1940) 

 concerning the presence of such a pigment as cytochrome c^. In addition, 

 Keilin and Hartree (1949) recognized the splitting of some of the absorption 

 bands of reduced cytochrome c giving rise to fine structure or what they 

 termed satellite bands. 



The use of low temperature spectroscopy is not new. Many varied types 

 of pigments such as porphyrins, nucleotides, chlorophyll and carotenoids 

 have been investigated at low temperature and the appearance of additional 



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