54 HEWSON SWIFT 



portance. In binding between fatty acids and protein basic groups, Boyer, 

 Ballou, and Luck^^ showed that the length of the carbon chain was an im- 

 portant factor. Larger molecules have increased binding energy, possibly 

 through their greater ability to release bound water molecules^^ although 

 the initial binding force is electrostatic. In studies on methyl orange-protein 

 binding, Klotz and Urquhart^' showed that buffer anion competition was 

 in general larger the larger the ion. Such factors are doubtless also of 

 importance in determining the dissociation constants of dye-nucleate com- 

 plexes. In some cases proteins have been shown to bind un-ionized com- 

 pounds, in which case nonelectrostatic forces must obviously be in- 

 volved.-^ '^^ Binding of this type may possibly be seen in nonspecific 

 adsorption of dye by tissue sections (see below). With some dyes, for example, 

 toluidine blue, this may be negligible after proper differentiation. Under 

 similar conditions, however, nonspecific adsorption of other dyes, such as 

 crystal violet, or basic fuchsin, may be intense. Van der Waals forces are 

 apparently responsible for the formation of dye complexes that occur in 

 more concentrated aqueous solutions of many dyes. This phenomenon is 

 associated with dye metachromasy, and is discussed below. 



1. Specificity 



As has frequently been pointed out, there are other acidic groups in 

 tissues besides nucleic acid, capable of binding basic dyes. Protein carboxyl 

 groups bind dyes at high pH. This has been shown, for example, by Singer 

 and Morrison^* in the case of fibrin films. Bartholomew, Evans, and Niel- 

 son" showed that basic dye binding was decreased at pH 9 and 11.5 when 

 carboxyl groups were blocked by methylation. Since the pK of protein 

 carboxyl groups is around 5, at pH levels below this carboxyl binding by 

 most basic dyes is negligible, for example, in tissue proteins after nucleic 

 acids have been removed by treatment in hot trichloroacetic acid or en- 

 zymes.^ -^^ This is also evident in Fig. 1, showing nucleic acid and protein 

 staining with azure B. In the pH range from about 3 to 5, basophilia in 

 most cases is due entirely to nucleic acid. Above pH 5, basophilia is also 

 due to carboxyl binding. 



The mucin of goblet cells, mast cell granules, and the chondroitinsulfuric 

 acid of cartilage are also strongly basophilic, presumably because such 



21 P. D. Boyer, C. A. Ballou, and J. M. Luck, J. Biol. Chem. 167, 407 (1947). 



" I. Klotz, Cold Spring Harbor Symposia Quant. Biol. 14, 97 (1950). 



" I. Klotz and J. M. Urquhart, J. Am. Chem. Soc. 71, 1597 (1949). 



" I. Klotz and J. Ayers, Federation Proc. 11, 240 (1952). 



" G. M. Allenby and H. B. Collier, Arch. Biochem. and Biophys. 38, 147 (1952). 



2« M. Singer and P. H. Morrison, J. Biol. Chem. 175, 133 (1948). 



" J. W. Bartholomew, E. E. Evans, and E. D. Nielson, J. Bacteriol. 58, 347 (1949). 



28 M. Flax and A. W. Pollister, Anat. Record 105, 56 (1949). 



