AfeGlNASE 507 



&tid the very weak base urea. By titration to a pH at which the 

 guanido group of arginine is completely ionized and the e-amino 

 group of ornithine (and urea) is not ionized, the cleavage of arginine 

 can be followed as the increase in the quantity of base required to' 

 bring the reaction mixture to this pH. The increase in the amount 

 of base will be one equivalent for each mole arginine hydrolyzed.- 

 This principle was employed by Linderstr0m-Lang et ctl. The propef 

 pH w^as obtained by titration in acetone-alcohol with thymol blue' 

 indicator. This method has the advantage of convenience and ra- 

 pidity; however, as the authors pointed out, it cannot be used if a 

 large amount of urease is present because the subsequent action of 

 the enzyme on the urea will increase the titration over the theoretical 

 value of one equivalent per mole arginine. The urease activity is rela- 

 tively low at the pH optimum of arginase, but the factor becomes ap- 

 preciable nevertheless if high concentrations of urease are present. 

 The titration of the ornithine set free is only about 95% of the theo- 

 retical, but this can be corrected if necessary by applying the factor 

 1.05 to the results. 



The other method reported is based on the measurement of the 

 ammonia formed when urease is allowed to act on urea set free by 

 the action of the arginase. One volume of 0.10 A'' base in this method 

 corresponds to one volume of 0.05 N base in the acetone-alcohol 

 titration method. 



Linderstr0in-Lang, Weil, and Holier Methods for Arginase 



A. Urease Method 

 SPECIAL REAGENTS 



Substrate Solution. (0.1 M arginine, pH 9.5). Dissolve 0.2104 g. 



arginine hydrochloride in 1.335 ml. 0.5 N sodium hydroxide and 



water to make a final vol. of 10 ml. 

 Urease-Buffer Solution. Prepare 100 ml. of a soln. containing 7 g. 



urease (Squibb) and 35 ml. 0.5 M phosphate buffer, pH 6.8 (mix 



equal vol. primary and secondary phosphate) . 

 40% Sodium Hydroxide. 

 0.3 N Hydrochloric Acid containing 10 ml. 0.04% bromocresol 



purple/50 ml. 

 0.1 N Sodium Borate. 



