Studies on Problems of Cytochrome c Oxidase Assay 275 



DISCUSSION 



Assay of Cytochrome c Oxidase 



Slater: A practical use of cytochrome c oxidase assays is to measure the distribution 

 of mitochondria in various fractions obtained by differential centrifugation. The 

 assumption is made that all the cytochrome oxidase is in the mitochondria, so that 

 the distribution of cytochrome oxidase in the various fractions is the same as the 

 distribution of the mitochondria. We do this by using very high concentrations of 

 cytochrome c, with />-phenylenediamine as reducing agent in 0-05 m phosphate buffer 

 and measure the O2 uptake manometrically. Do you think that the inhibitor which you 

 find in the soluble fraction of tissue homogenates causes serious error in this procedure ? 



Smith : Yes, I do. Like other workers, we have found that the sum of oxidase activity 

 from different fractions of a tissue homogenate is usually considerably larger than the 

 activity of the unfractionated homogenate. 



Lemberg : The paper of Smith shows how important it would be to have an analytical 

 method for the estimation of the haem a content of tissues without reference to enzyme 

 activity. 



I mention only one example. Lahey, Gubler, Chase, Cartwright and Wintrobe 

 {Blood 7, 1053, 1952) had believed that the lack of cytochrome c oxidase activity in 

 organs of copper-deficient swine was due to the lack of copper in the oxidase molecule. 

 However, Gallagher, Judah and Rees {Proc. roy. Soc. 5145, 134, 1956) have shown 

 that haem a was almost completely absent from the liver in copper-deficient rats. 

 Since Lahey et al. {loc. cit.) had found no lack of catalase in copper-deficient swine, 

 it is evident that copper is specifically required for the biosynthesis of haem a from 

 protohaem or its precursors. 

 Three such analytical methods have been worked out by us : 



(1) The quantitative isolation of porphyrin a in a state of spectroscopic purity or 

 near-purity. This can be satisfactorily applied only to tissues comparatively rich in 

 haem a such as heart and requires at least 5 g of tissue. It is also technically not 

 quite easy. 



(2) The spectrophotometric determination based on measurements of the optical 

 densities at 587 and 558 m/t of the mixed pyridine haemochromes. This requires less 

 material but still a relatively high haem a content. 



(3) The separation of haemin a from protohaemin by a modified Rawlinson-Hale 

 procedure, followed by spectrophotometric analysis of the pyridine haemochrome. 

 This method has given satisfactory results in the study of iron incorporation into 

 haem a in rat tissues (Lemberg and Benson, Nature, Lond. 183, 678, 1959). The 

 presence of lipid in the extracts still causes some difficulties which we hope to overcome. 



Wainio: Suppose a surface active agent was added to the particulates presented in Table 



5 (Smith, p. 269), i.e. those that have different oxidase activity/AE 605-630 m/« values, 



would the results be more uniform? 

 Smith: Addition of low concentrations of cholate to heart muscle particles will increase 



the oxidase activity/AE 605-630 m/t almost up to that obtained with liver particles. 



We have not made observations with preparations from kidney or brain. 



Inhibition of Cytochrome c Oxidase by Cytochrome c 



Slater: Ever since Smith reported inhibition of the cytochrome c oxidase reaction by 

 oxidized and reduced cytochrome c, we have attempted to incorporate this inhibition 

 in reaction mechanisms, i.e. we thought that the inhibition in some way might be a 

 part of the normal mechanism. Am I correct in concluding that you regard the 

 inhibition as incidental to the enzymic reaction, i.e. that it is a side reaction caused 

 by the fact that cytochrome c is a highly basic protein? If this is so, perhaps we have 

 been wasting our time, and simpler mechanisms will be adequate. 



Smith: I feel that the observation that the inhibitory effect of cytochrome c can be dupli- 

 cated by another basic protein, salmine, fits better with the assumption that the inhibi- 

 tion is a side reaction. Also in accord with this view is the lack of inhibition of electron 



