l8 NITROGEN METABOLISM 



(e.g. phenosafranine and benzyl viologen, but not methy- 

 lene blue) and thus restore the original dye colour. Proline, 

 hydroxyproline and glycine were reduced by such a system, 

 though ammonia was only formed from glycine. Incubation 

 of alanine with proline resulted in the production of i mole 

 of NH3 per mole of alanine: none was produced from the 

 separate amino-acids. Stickland thus demonstrated that CI. 

 sporogenes catalysed oxido-reduction reactions between pairs 

 of amino-acids, one acid acting as a H-donor, the other as 

 a H-acceptor. The reduction of proline resulted in opening 

 of the ring and the formation of (5-aminovaleric acid, whilst 

 the products derived from alanine were NH3 , CO 2 and 

 acetic acid. When cresyl blue accepted hydrogen from 

 alanine, 2 moles of dye were decolorized for each mole of 

 NH3 released, indicating that the overall oxidation of 

 alanine involved the donation of four hydrogen atoms. 

 Although there was no direct evidence, it seemed highly 

 probable that pyruvate was an intermediate in the decompo- 

 sition of alanine and Stickland therefore proposed that the 

 overall reaction {d) represented the sum of three separate 

 reactions (a, b, c): 



CH3CH(NH2)COOH+H.30=NH3+CH3COCOOH+2H (a) 

 CH3COCOOH+H20=CH3COOH+C02+2H (b) 



4H+2NH(CH2)3CHCOOH=2NH2(CH2)4COOH (c) 



CH3CH(NH2)COOH+2NH(CH2)3CHCOOH+2H20 



=2NH,(CHo)4COOH+NH3+C02+CH3COOH (d) 



similarly: 



CH3CH(NH2)COOH+2NHoCH,COOH+2H20 



=3NH3+C02+3CH3COOH 



Clostridium botiilinum [15] and all the proteolytic Clos- 

 tridia examined by Nisman, Raynaud and Cohen [45] were 

 capable of performing the Stickland reaction; amongst the 

 organisms which could not were CI. tetani, CI. tetano- 

 morphum, CI. welchii and CI. saccharobutyricum. Apart from 

 the substances already mentioned, histidine, serine, iso- 



