Enzymes 193 



1) The lead method."'^— Composition of the substrate mix- 

 ture: In 500 ml. of a 0.05 M acetate buffer of pH 5 dissolve 

 0.6 g. of lead nitrate (about 0.003 M) and add 50 ml. of a 

 3 per cent (about 0.1 M) solution of Na glycerophosphate. 

 The mixture will become turbid; the degree of turbidity de- 

 pends on the percentage of y8-isomer contained in the brand 

 of glycerophosphate used. A mixture of about equal parts 

 of the two isomers will cause much less turbidity than the 

 more commonly sold mixtures containing around 75 per cent 

 of y8-salt. Keep the solution in the incubator at 37° C. for 

 24 hours, filter it. Add a small amount (about 5 per cent) of 

 distilled water to the filtrate to prevent precipitation on 

 evaporation. The mixture is ready for use and will keep in 

 the icebox for months. If it becomes turbid, it should be 

 discarded. 



Carry sections through xylene and alcohols to water. Col- 

 lodion coating of the sections is advisable; it appears to pre- 

 vent loss of enzyme by diffusion (Doyle^^), especially at 

 sites of low activity, although it may cause some patchiness 

 of the reaction ( Goetsch and Reynolds"^^ ) . Rinse slides thor- 

 oughly in distilled water. 



Incubate in the substrate mixture around 37° C. for 1-24 

 hours. Human prostate usually requires 1-^2 hours of incuba- 

 tion, other tissues 6-S hours or more. Sites of activity will 

 become a chalky white from the deposition of lead phos- 

 phate. Rinse slide first in distilled water, then for a minute 

 or so in 1-2 per cent acetic acid and once more in distilled 

 water. Immerse slide in a dilute solution of ammonium sul- 

 fide (a few drops to a Coplin jarful of distilled water) for 

 about 2 minutes. Wash under the tap. Counterstain as de- 

 sired. Dehydrate in alcohols; clear in gasoline or tetrachloro- 

 ethylene, and mount in clarite or some similar resin dissolved 

 in the same solvents. Toluene or xylene should not be used; 

 they will cause some fading of the stain. Sites of activity are 

 indicated by the dark brown -black precipitate of PbS. 



76. Gomori, G.: Stain Techno!., 25:81, 1950; Wang, K. J.: Chinese J. 

 Physiol., 17:317, 1950. 



