Studies on Problems of Cytochrome c Oxidase Assay 279 



Wainio: In 1951 we demonstrated that the oxidation of ferrocytochrome c by cytochrome 

 c oxidase is maximal at pH 6-0 in 0- 1 m phosphate buffer and that the activity decreases 

 sharply on both sides of the optimum (/. biol. Chem. 192, 349, 1951). More recently 

 (unpublished) we have extended these studies, and although it appears as though the 

 charge on the cation may be the controlling factor, there is still some question as to 

 whether the total ionic strength is not also important. We are investigating the 

 possibility that the increase in activity as the concentration of the buffer is raised 

 from zero may be determined by the total ionic strength and that the decrease after 

 the optimum may be largely an action of the cation charge on the A',,,, as set forth by 

 Estabrook. 



The extinction coefficient (reduced-oxidized at 605 m/O which we used to calculate 

 the haem content was obtained in our experiments where we used ferrocytochrome c 

 as a reductant {J. biol. Chem. 216, 593, 1955). Under anaerobic equilibrium conditions 

 the moles of ferrocytochrome c oxidized were assumed to equal the gram atoms of 

 iron (or haem) of cytochrome c oxidase that were reduced. 



From the haem: protein ratio of 7-0 which is given in Table 1 it may be calculated 

 that cytochrome c oxidase has a molecular weight of 140,000. However, this is based 

 on the assumption that the total nitrogen of Fraction 6 (cytochrome c oxidase N + 

 other protein N + lipid N) is cytochrome c oxidase nitrogen. These fractions have 

 at no time been represented as being pure preparations of cytochrome c oxidase. 

 The fractionation technique was applied only for the purpose of obtaining haem:Cu 

 and Cu: activity ratios. The total protein was simply the common denominator before 

 the ratios were taken. 



HoRio: I should like to make a comment concerning the effects of native and modified 

 cytochrome c on the oxidase activity, about which Henderson asked a question. Of 

 the various modified cytochromes c preparations that we have found, at least one 

 exists in a pure dimer form. Its molecular weight is calculated to be 24,400 based on 

 its sedimentation and its diffusion coefficients. Compared with the native monomer 

 form, this dimer form activates both the purified cytochrome a preparation and the 

 particulate oxidase preparation very little. 



Margoliash: In view of Smith's findings and those that Estabrook has just referred to 

 in discussion, on the effect of cytochrome c and salmine as well as that of inorganic 

 cations on the cytochrome oxidase reaction, would it not be possible to consider the 

 binding of cytochrome c to the oxidase as due to a polycation-polyanion type of 

 electrostatic interaction ? I think Estabrook's description of the effect of polyvalent 

 cations particularly suggestive in this respect. The extreme basicity of mammalian 

 heart cytochrome c as well as Morrison's finding of an acid isoelectric point for 

 cytochrome oxidase preparations would also fit such an interpretation. 



Boardman: The interaction between a protein such as cytochrome c and a polyanion 

 appears to be very complex. Some of the factors governing such an interaction have 

 been elucidated by studying the adsorption of cytochrome c on a polymethacrylic 

 acid ion-exchange resin. The adsorption of cytochrome c on the resin is very depen- 

 dent on the cation concentration and the pH of the medium. But it does not seem 

 possible to explain the experimental data by visualizing the interaction as a purely 

 electrostatic one between a polyvalent cation and a polyvalent anion. With substances 

 of high molecular weight, short range forces such as Van der Waals or hydrogen 

 bond forces may play an important role in the adsorption. 



However, a study of the relationships between cytochrome c concentration, cation 

 concentration and pH on the one hand and oxidase activity on the other does seem 

 to provide an experimental approach to the problem of what is happening in the 

 cytochrome c oxidase complex. 



Armstrong : There is a cytochrome c in Micrococcus denitrificans which has an isoelectric 

 point of less than pH 7, which might be used to test this theory. 



Slater : Horio refers in his paper to the fact that his value for the A",,, for cytochrome c 

 in the system reducing agent — cytochrome c-oxidase-oxygen is the same as ours. It 

 is perhaps desirable to point out that, if we accept Smith and Conrad's interpretation 

 of the inhibitory action of cytochrome c on cytochrome oxidase, this Ar„, actually 



