106 



A SYMPOSIUM ON RESPIRATORY ENZYMES 



The method that led to the isolation of codehydrogenase I in a pure 

 state is based in the initial stage on the purification steps recom- 

 mended by Myrback (le). A definite improvement is the precipita- 

 tion of cozymase by cuprous chloride dissolved in a concentrated 



Table 2.— Preparation of codehydrogenase II from horse erythrocytes 

 WASHED ERYTHROCYTES 



hemolysed by water; 

 precipitated with acetone 



SOLUTION 



acetone removed by 

 vacuum distillation; 

 fractional precipitation by Hg(0Ac)2 



PRECIPITATE (protein) 

 (discarded) 



FRACTIONS 2 and 3 (contain Co II) 



+H2S (HgS i ) 

 +acetone 



FRACTIONS 1 and 4 

 (discarded) 



PRECIPITATE (contains Co II; purity about 15%) 

 dissolved in H2O; 



fractional precipitation by Ba(0H)2 

 -f alcohol (separation from Co I and cophosphorylase) 

 -|-Hg(0Ac)2; precipitate +H2S; 

 solution -{-acetone 



PRECIPITATE (contains Co II; purity about 30%) 

 dissolved in HCI-CH3OH; 

 precipitation by ethylacetate 



CODEHYDROGENASE II (purity about 50%) 



fractional precipitation by lead acetate-}- alcohol 



FRACTION 1 (about 60%) FRACTION 2 (about 60%) 



FRACTIONS 3 

 and 4 (about 100%) 



solution of potassium chloride (9). This procedure has been em- 

 ployed in all preparative methods subsequently recommended 

 (10-13). Since pure cozymase does not give a stable precipitate with 

 the cuprous chloride reagent, such precipitation cannot be repeated. 

 Apparently some impurity in the crude cozymase solutions plays an 

 important role in producing a stable precipitate. Decomposition of 

 the precipitate by hydrogen sulfide involves a considerable loss of 



