THIAMINE AND COCARBOXYLASE 395 



when more than 0.01 /tg. cocarboxylase was present; however. 0.001 

 fig. proved to be a favorable amount. 



There is little reason why the Cartesian diver could not be applied 

 to the method of Atkin et al. (1939) and Schultz et al. (1942), which 

 depends on the stimulation of yeast fermentation by thiamine. 



Westenbrink Method for Thiamine and Cocarboxylase 



SPECIAL REAGENTS 



Yeast Suspension. Stir 100 mg. yeast with 5 ml. 0.1 M secondary 

 sodium phosphate for 4 min. at 16-20°. Centrifuge 1 min.; dis- 

 card supernatant and repeat the procedure twice. Finally wash 

 residue once with 5 ml. water in the same manner. To the residue 

 now add 0.12 ml. of 0.1 M magnesium chloride and enough 0.1 M 

 phosphate buffer, pH 6.2, to bring the total vol. to 1.2 ml. Use soon, 

 since the treated yeast deteriorates rapidly even in the cold. 



1.0 M Sodium Pyruvate in 0.1 M phosphate buffer, pH 6.2. 



Thiamine (0.8 mg. per ml.) in 0.1 M phosphate buffer, pH 6.2. 



Cocarboxylase (0.005 mg. per ml.) in 0.1 M phosphate buffer, pH 

 6.2. 



PROCEDURE 



1. Pipette into a Cartesian diver, having a vol. of 20-30 lA., 0.2 /il. 

 portions of the pyruvate, thiamine and cocarboxylase solns. When 

 thiamine is to be determined, use the cocarboxylase soln. with 0.2 

 /xl. unknown; and when cocarboxylase is to be measured use the 

 thiamine soln. with the unknown. 



2. Set up a control experiment by replacing the unknown soln. 

 by the phosphate buffer. 



3. Place divers containing the reagents in glass tubes closed by 

 rubber stoppers to prevent evaporation. 



4. Prepare the yeast suspension and add 0.5 /xl. of it to the solns. 

 in the divers. 



5. Seal the neck of each diver with 1.7 /xl. mineral oil; place diver 

 in the apparatus and take readings of the gas evolution after 30 min. 



6. Calibrate the measurements by comparisons with those ob- 

 tained using known amounts of the constituent being analyzed. 



