512 



R. K. Morton, J. McD. Armstrong and C. A. Appleby 



acids (Table 5). Of the compounds tested, lactate is clearly the most effective 

 substrate. Table 5 also shows that a number of other hydroxy acids strongly 

 inhibit oxidation of lactate; D-malate is particularly inhibitory. Neither 

 reduced di- or tri-phosphopyridine nucleotide (Yamashita et al., 1957; 

 Yamanaka et al, 1958) nor malate (Yamanaka et «/., 1958; Yamashita et al, 

 1958) are substrates of the crystalline enzyme. 



The results show that there is a high degree of specificity at the substrate 

 binding site. 



Table 5. Substrate specificity of crystalline cytochrome 6, 



Relative activities were determined spectrophotometrically by the rate of reduction 

 of potassium ferricyanide at pH 80 in 033 m sodium pyrophospliaie-HCl buffer 

 containing 10^^ m EDTA. Relative inhibition was determined under generally 

 similar conditions with DL-sodium lactate (01 m) as substrate. Where appropriate 

 the DL-compounds were used in all cases, unless otherwise specified, at OT m 

 concentration. Results of Armstrong and Morton (1959). 



1 D-malate (commercially prepared material), 001 m con- 

 centration. 



2 L-malate (commercially prepared material), 005 M con- 

 centration. 



^ L(+)-tartrate and L(+)-a-hydroxy-glutarate, 0-05 M con- 

 centration. 



Reactivity of Crystalline Cytochrome h^ with Oxygen, Ferricyanide, Cyto- 

 chrome c and Dyes 

 Crystalline cytochrome />2 containing very small amounts of lactate is 

 slowly oxidized aerobically. At pH 7-4 and at 0°C in the presence of 0-5 m 

 NaCl, 0-1 mM EDTA, and 001 m Tris (2-amino-2-hydroxymethylpropane- 

 1 : 3-diol)-HCl buffer, the rate of oxidation was equivalent to the oxidation 



