PEPTIDASE 419 



as a maximal time for measm-ements, and the density determined to 

 1 X 10"■^ with 0.1 fA. drops, the least quantity of alanylglycine split- 

 ting that could be detected in 10 hours would be 1 X 10""^ millimole. 

 This splitting could be accomplished by one thousandth of one sea 

 urchin egg, corresponding to about 5 X 10"^ mg. dry organic mate- 

 rial. 



Jacobsen (1942) showed that the contractions accompanying the 

 cleavages of peptide linkages in benzoyl-DL-argininamide by trypsin 

 and benzoyl-L-tyrosylglycinamide by chymotrypsin are 13.4 ± 0.2 

 and 15.6 ± 0.8 ml. per mole, respectively. 



Method of Linderstr^ni-Lang and Lanz for Peptidase 



SPECIAL REAGENTS 



Enzyme Preparation. 60% glycerol extract of tissue diluted with 



phosphate buffer, pH 6.8 to 7.4, or a bit of cellular material can be 



used directly. 

 Substrate Solution. 0.2 M OL-alanylglycine containing sodium 



hydroxide of the molarity indicated in Table X to give the desired 



pH. 

 Mixture for Saturation of Medium. Combine equal vol. of 0.2 M 



potassium bromide and substrate soln. 



PROCEDURE 



1. Saturate the medium in the gradient tube with the potassium 

 bromide-substrate soln. mixture in the manner described in the 

 preceding section. After 24 hr. the tube may be used. 



2. Introduce a number of 0.15 fA. standard drops into the tube. 

 The sp. gr. of these drops should bracket the range encountered in the 

 determination. Two drops of the same standard should not deviate 

 from one another in their equilibrium levels by more than 0.1 mm. 



3. Mix equal vol. enzyme and substrate soln. under kerosene in 

 a small test tube, and place 2-3 drops 0.15 ix\. each in the tube. For 

 microtome sections of tissue, or smaller cellular units, first place a 

 1 ix\ kerosene drop on the end of a glass needle about 0.2 mm. 

 thick; insert the tissue into this drop with the aid of a very fine glass 

 needle, dip the drop containing the tissue into the gradient tul)e 



