NEW HISTOCHEMICAL METHODS 



FOR THE DEMONSTRATION 



OF PROTEIN-BOUND TRYPTOPHAN 



AND TYROSINE: 



A CRITIQUE AND EVALUATION 



G. G. GLENNER 



National Institutes of Health, Bethesda, Maryland, U.S. A. 



Within the last three years the introduction of new histochemical tech- 

 nicpies for the démonstration of protein-bound tryptophan and tyrosine hâve 

 niade possible sensitive, spécifie and permanent localization of proteins 

 containing thèse amino acids in tissue sections. Because of their high 

 specificity, thèse new histochemical methods hâve elevated to a promi- 

 nent position the démonstration of proteins in tissues by means of their 

 amino acid constituents. 



With the benzylidene condensation reaction as thebasis for the démon- 

 stration of the indole ring, two important methods for the localization of 

 protein-bound tryptophan in tissue sections hâve been established. The 

 first of thèse is the "post-coupled benzylidene" reaction and the second, 

 the "rosindole" or "carboline" reaction. The "post-coupled benzylidene" 

 reaction makes use of a freshly prepared diazonium sait to increase the 

 color of the initial benzylidene condensation product by a post-coupling 

 reaction. The "rosindole" or "carboline" reactions utilize an oxidant 

 to produce a stable blue dye from the initial benzylidene product. It ap- 

 pears that the only différence between the "rosindole" and "carboline" 

 reactions is the solvent and proportion of condensing agent to solvent 

 in the benzylidene reaction médium. However, this différence in solvent 

 and solvent condensing agent ratio appears to affect the reaction sensi- 

 tivity. From expérimental évidence it appears that in demonstrating 

 sites not shown by the "carboline" method, the "rosindole" technique 

 is solubilizing less protein from tissue sections and, therefore, appears 

 to be more sensitive. 



The methods for protein-bound tyrosine dépend on the nitrosation of 

 tyrosine with simultaneous chelation of the ortho nitrosotyrosine with 

 various cations to produce a color reaction. In the absence of thèse 

 cations further exposure to nitrous acid produces an ortho-diazotyrosine 



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