532 E. BOERI AND M, RiPPA 



is blocked by increasing the lactate concentration. As metal ions increase the 

 oxygen consumption, the obvious explanation seems that lactate binds the 

 metal ions and therefore inhibits the autoxidation. 



Figures 9 and 10 show that this explanation is correct. Among metals 

 tested, iron was found the most active catalyser. Less active were cobalt 

 (C0CI2), manganese (MnClg) and molybdenum (Na2Mo04). 



Copper (CUSO4) strongly inhibited the autoxidation. 



The effects of o-phenanthroline were difficult to interpret. It was expected 

 that o-phenanthrohne, as a complexing agent for bivalent iron, would 

 remove the effect of this metal. However, it was found that when o-phenan- 

 throhne and ferrous ions were added, the effect depended on the ratio of the 

 two reagents. With a ratio of 1:1, the catalytic effect on the autoxida- 

 tion is even higher than with ferrous ions alone. It is known that o-phenan- 

 throline is a tridentate chelating agent and binds ferrous ions up to formation 

 of the complex Fe"-(o-phenanthroline)3. When this complex was tested, it 

 was still found to be catalytically active on the autoxidation rate, although 

 less active than ferrous iron alone. Actually, this fact appears less strange 

 when we think that a similar compound, ferricyanide, which also has six 

 valences of iron blocked in a diamagnetic compound, is very accessible to 

 flavocytochrome b^, being the acceptor of choice for testing its activity 

 (Appleby and Morton, 1954). 



Finally, it must be remembered that high amounts of non-haematin iron 

 accompany the enzyme during its purification, only the last step removing 

 the last traces of it. This seems the reason why the unpurified enzyme appears 

 much more autoxidizable than purified preparations. Our present prepara- 

 tions have only traces of non-haematin iron. 



Factors which favour the autoxidation (high oxygen and metal and low 

 lactate concentrations) tend to inactivate the enzyme. We have computed 

 from activity analysis that the enzyme has lost about half its activity when it 

 has cycled 750 times with oxygen as the acceptor. The decay is infinitely less 

 when ferricytochrome or ferricyanide are the acceptors. 



According to Morton (1958), the fact that removal of heavy metals and 

 oxygen protects the enzyme is in favour of a bond between — SH groups of 

 certain of the cysteine residues of the protein and the imino group of 

 the riboflavine phosphate. The present results are in favour of such an 

 explanation. 



The effect of ionic strength should be compared to the effect shown when 

 other oxidants are used. When ferricyanide is the oxidant, there is no effect 

 due to the ionic strength. When cytochrome c is the oxidant, high ionic 

 strength inhibits the reaction. With oxygen, instead, there is an optimal ionic 

 strength. 



In conclusion, as noted by Morton (1958), flavocytochrome b^ is a very 

 slightly autoxidizable substance. The complete inhibition of autoxidation 



