86 



K. Kaziro and K. Tsushima 



(Tsushima and Miyajima, 1956). Here we wish to mention one more experi- 

 ment which was done with/7-chloromercuribenzoate (PCMB) (Kajita, 1956b). 

 As aheady reported by Riggs (1952), the haem-haem interaction of haemo- 

 globin decreases on addition of PCMB. We have observed, however, that the 



5-5 



50 4-5 



— logCPCMB), M 



3-5 



Fig. 5. Effect of /»-chloromercuribenzoate on the oxidase activity of haemoglobin 

 and myoglobin. Haemoglobin and myoglobin concentration, 3 x 10~^ M, ascor- 

 bate concentration, 2-5 x 10~^ m. The measurements were made at pH 7-5 and 



at 37°C. 



addition of PCMB also gives rise to a marked increase in the oxidase activity 

 of haemoglobin. As shown in Fig. 5, the oxidase activity of haemoglobin 

 increases with the increase of PCMB concentration as compared to that of 

 native haemoglobin. The maximum oxidase activity of haemoglobin (indi- 

 cated by an arrow in Fig. 5) can be reached by the addition of two molecules 

 of PCMB per atom of haemoglobin-iron. 



In the study with cytochrome c, we took the appearance of the absorption 

 spectrum of the CO-compound as one of the criteria of modification, taking 

 advantage of the fact that native cytochrome c does not bind CO. As shown 

 in Fig. 6, cytochrome c becomes able to bind CO at a concentration range of 

 benzoate of 1-6-2-0 m. A similar relation obtains in experiments with 

 salicylate. The addition of benzoate also makes cytochrome c active as 

 oxidase. The oxidase activity of cytochrome c increases with increased 

 addition of benzoate. The activity decreases, however, when the concentra- 

 tion of benzoate exceeds 1-0 m, suggesting that a progressive stage of modifica- 

 tion of cytochrome c is brought about by the addition of higher concentrations 

 of benzoate. Progressive modification of the cytochrome c molecule was 

 found to be favourable to the formation of the CO-cytochrome c complex. 

 The stepwise modification of the cytochrome c molecule appears to be similar 

 to that which was observed with haemoglobin. It is noteworthy, however, 

 that the oxygen activating ability of cytochrome c (using ascorbic acid as 

 substrate) is brought about by lower concentrations of benzoate than the 



