Enzymes 217 



devised two methods to avoid the latter difficulty: (1) 8- 

 hydroxyquinoline glucuronide is used as a substrate, and 

 8-hydroxyquinoline is precipitated as its ferric salt; (2) o- 

 aminophenol glucuronide is prepared first by biosynthesis, 

 then diazotized, and coupled with /3-naphthol. In this way 

 the glucuronide of y8-naphthylazo-o-phenol is obtained. This 

 is a red dye, slightly soluble in water, while the phenol it- 

 self, liberated from it by enzymatic action, is insoluble and 

 will precipitate. Sehgman and co-workers^^^ object to the 

 use of a colored substrate, fearing nonenzymatic staining 

 effects. They propose the use of glucuronides of highly in- 

 soluble naphthols, such as 6-bromo-y8-naphthol or 8-ben- 

 zenesulfonamido-y8-naphthol; incubation is to be carried 

 out in the absence of a diazonium salt, and azo-coupling of 

 the precipitated naphthol is performed in a second step. No 

 reports have been published so far on the actual use of this 

 substrate. As pointed out before (p. 171), this type of tech- 

 nique is not likely to work out satisfactorily. 



Since the substrates are not available commercially and 

 are almost impossible for most workers in the biological 

 fields to prepare, only the essential features of the Frieden- . 

 wald-Becker methods will be given here. 



Fresh-frozen sections are used; the substrate is buffered 

 with acetate at pH 5. 



1. Incubate tissues in a saturated solution of y8-naphthyl- 

 azo-o-phenyl glucuronide for 1-5 hours. Wash sections, fix 

 them in formalin, and counterstain with hematoxylin. Mount 

 in glycerol jelly. Sites of activity are briUiant red; back- 

 ground, orange-yellow. 



2. Saturate a solution of 8-hydroxyquinoline glucuronide 

 containing 0.03 N ferric chloride with ferric hydroxyquin- 

 ohne. This saturation of the incubating medium with the 

 compound to be produced enzymatically is essential. Incu- 



131. Seligman, A. M., Nachlas, M. M., Manheimer, L. H., Friedman, 

 O. M., and Wolf, G.: Ann. Surg., 130:333, 1949; Seligman, A. M., and 

 Nachlas, M. M.: Cancer Research, 10:240, 1950. 



