Enzymes 183 



(ferrous sulfate followed by f erricyanide ) . Boume^^ suggests 

 staining with alizarinesulfonate. 



A very interesting method of visualization has been de- 

 scribed by Dalgaard.^^ If glycerophosphate containing radio- 

 active phosphorus is used, the precipitate can be demon- 

 strated by its radioautogarph. The secondary introduction of 

 radioactive lead into the precipitate is also possible.*^ 



The sensitivity of the method, as determined by the Cou- 

 jard method, is about 20-30 ^LtM -units (0.5-1 Bodansky unit) 

 per gram of active structure, the length of incubation being 

 1 hour. Proportionahty holds for the period of between 2-3 

 minutes and 12 hours. 



Sources of error.^^— 



a) False negative reactions (i.e., negative reactions in 

 spite of the chemically verified presence of enzyme ) will be 

 obtained whenever the concentration of the enzyme is below 

 the threshold of sensitivity of the method. Inactivation of the 

 enzyme by technical error is an unlikely complication, pro- 

 vided that the method suggested is adhered to with reason- 

 able accuracy. However, if for special reasons experimental 

 conditions have to be changed considerably (use of low pH 

 values, inhibitors, high ionic strength ) , negative results must 

 be interpreted with caution. 



b) False positive reactions were briefly mentioned on 

 page 148. Pigments can be seen in unincubated sections; 

 some of them can be identified by specific reactions (hemo- 

 siderin, ceroid, etc.). Preformed calcifications which will 

 give reactions indistinguishable from true enzymatic ones 

 can be removed before incubation by treating the sections 

 for about 10 minutes with a citrate buflFer of pH 4.5-5. The 

 adsorption of cobalt salt on tissue protein is not likely to 

 cause any trouble. Artifacts due to the spontaneous or bac- 



45. Bourne, G.: Quart. J. Exper. Physiol., 32:1, 1943. 



46. Dalgaard, J. B.: Nature, 162:811, 1948. 



47. Barka, T., Szalay, S., Posalaky, Z., and Kertesz, L: Kiserletes orvos- 

 tud., p. 1, 1951. 



48. Gomori, G.: J. Lab. & Clin. Med., 35:802, 1950. 



