CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 



57 



PH 



■% 



Fig. 2. The influence of proteins on basic dye binding. A. Dye/phosphorus ratio 

 in precipitate from 1 ml. of 0.1% solution of PNA added to 9 ml. of KT* M toluidine 

 blue. B. Same, with 0.1% fibrinogen added. C. Dye/phosphorus ratio in muscle of 

 17-day rat embryo stained with 10"^ M toluidine blue. Protein basic groups depress 

 dye bending at lower pH; carboxyl binding is apparently responsible for the increased 

 staining at pH 6.8. D. Orange G (10~* M) binding by fibrin films, as an indication of 

 the increasing charge of protein basic groups with decreasing pH. [A, B, and C from 

 Herrmann, Nicholas, and Boricious*; D from Singer and Morrison'' '.] 



presence of protein basic groups has apparently decreased the basic dye 

 adsorption. As one indication of the increased charge with decreasing pH 

 shown by protein basic groups, the data of Singer and Morrison-^ for orange 

 G binding of fibrin fihns is shown in curve D. This curve is roughly charac- 

 teristic of a wide variety of proteins. The increase of acid dye binding with 

 decrease in pH might not be expected, since the amino, guanrdyl, and 

 imidazole groups should be maximally ionized below pH 7. Apparently 

 these groups are not always free to react, possibly because they are blocked 

 by internal hydrogen bonding between amino and hydroxyl or carboxyl 

 groups in the protein molecule, as suggested by Klotz.^^ 



The equilibrium between unbound dye and the dye bound to a tissue 

 section is thus influenced by pH through its action on the total electrostatic 

 charge on the nucleoproteins of the tissue. Other factors can obviously shift 

 the equilibrium in one direction or another. The role of these variables in 

 textile dyeing has been discussed by Vickerstaff,^* and their influence, 

 largely as applied to protein binding, has been reviewed by Singer.'* 



" T. Vickerstaff, "The Physical Chemistry of Dyeing". Imperial Chemical In- 

 dustries, London, 1950. 

 »8M. Singer, Intern. Rev. Cytol. 1, 211 (1952). 



