DIFFUSION OF ENZYME DURING FIXATION 47 



Diffusion of Enzyme during Fixation 



We have seen that the position of an enzyme within a section 

 may be determined with some accuracy and that the extent 

 to which the enzyme diffuses within a section may also be de- 

 termined without difficulty. It remains to be seen whether 

 the enzyme present in a fixed preparation is in its physiologically 

 normal position. 



As was indicated in the chapter on fixation, the only method 

 of fixation which is sufficiently rapid to exclude diffusion of an 

 enzyme is freeze-drying. Where this method is used, one may 

 be sure that diffusion of phosphatase does not occur during fix- 

 ation. Where freeze-drying cannot be used, the extent of dif- 

 fusion, and the site at which the diffused enzyme appears, can 

 in some cases be detected, provided that a wide variety of fixa- 

 tives are used. With a variety of fixatives the time avail- 

 able for diffusion and the adsorption conditions prevailing in 

 the specimen should vary considerably from fixative to fixative. 



In a study of the action of chemical fixatives, a number of in- 

 dividual components of fixatives were tested for their direct ac- 

 tion upon the enzyme by placing a section from an alcohol- 

 fixed block in the fixative component. After 2 hours' exposure 

 to the fixative component the sections were washed carefully 

 and then incubated for the same length of time as control sec- 

 tions. The fixative components tested in this way were 8 per- 

 cent formaldehyde; saturated picric acid; saturated magnesium 

 sulfate; saturated ammonium sulfate; saturated calcium chlo- 

 ride; saturated mercuric chloride; 1 percent, 5 percent, 10 per- 

 cent, and 50 percent acetic acid; 1 percent trichloroacetic acid; 

 10 percent pyridine; acetone; chloroform; dry pyridine, alco- 

 holic iodine, 1 percent osmic acid; 1 percent iodine in potassium 

 iodide; 0.04 percent reduced thioglycollate; 0.04 percent re- 

 duced glutathione; and 0.04 ^percent oxidized glutathione. 

 Phosphatase was completely destroyed by mercury, osmic acid, 

 trichloroacetic acid, iodine, and concentrations of acetic acid 

 higher than 1 percent. There was a marked reduction in the 

 amount of enzyme activity caused by formaldehyde, magnesium 

 sulfate, ammonium sulfate, picric acid, and reduced thiols, but 

 the amount of reduction in activity was not sufficient to preclude 

 their use in fixatives. 



