PROTEOLYTIC ENZYMES 303 



of tissue, have been developed by the Carlsberg Laboratory in- 

 vestigators. Linderstr0m-Lang and Holter (1932) described a 

 method for dipeptidase based on the acetone titration of amino 

 groups. AVith DL-alanylglycine as the substrate, the procedure has a 

 ])recision of about 0.08 /A. of 0.05 A^ hydrochloric acid corresponding 

 to 5.6 X 10"^ mg. amino nitrogen. Holter and Linderstr0m-Lang 

 ( 1932 1 extended the method to include proteases of both the peptic 

 and catheptic type. The maximum deviation in this case amounts 

 to about 0.16 ix\. 0.05 N acid or 1.2 X 10"^ mg. amino nitrogen. By 

 substitution of the appropriate substrate, the acetone titration micro 

 method has also been used for aminopolypeptidase, carboxypoly- 

 peptidase and prolinepeptidase by S0eborg-Ohlsen (1941) and 

 others. Buffers need not be added in these measurements, since the 

 substrates have sufficient buffer capacity of their own. 



The alcohol titration for carboxyl groups was adapted by Linder- 

 str0m-Lang and Duspiva ( 1936) for the alkalimetric measurement 

 of protease; their method has a precision of about 0.3 ix\. 0.05 N 

 alkali. Because of precipitation, solutions more acid than pH 8 can- 

 not be titrated by this procedure. 



Weil ( 1936) described a formol titration for the determination of 

 tryptic activity on a similar scale, which he reported to be repro- 

 ducible to ±0.1 fA. 0.05 N alkali. 



The dilatometric method for peptidase is described on page 417. 



Linderstr^m-Lang and Holter Acidimetric Acetone Method 

 for Proteolytic Enzymes 



SPECIAL REAGENTS 



Enzyme Extraction Mediujn. 30 ml. 88% glycerol + 5 ml. M/15 

 primary potassium phosphate -f- 5 ml. M/15 secondary sodium 

 phosphate made up with water to 100 ml. 



Acetone Containing 2 Milligram Per Cent Naphthyl Red. 



0.05 N 90% Alcoholic Hydrochloric Acid. 



Substrates. The peptidase substrates are 0.2 M solns. of the racemic 

 peptides containing sodium hydroxide to give the desired pH, e.g., 

 0.036 M alkali gives a pH of 7.4 to 0.2 M alanylglycine. An excep- 

 tion is leucylglycine which must be used in 0.18 M soln. because 

 of its sparing solubility. 

 Dipeptidase. Alanylglycine or leucylglycine. 



