56 HEWSON SWIFT 



run, in which nucleic acid has been removed with ribonuclease, deoxyribo- 

 nuclease, or acid treatment. These methods should distinguish other types 

 of binding, such as that due to sulfuric acid esters, phosphoproteins, or the 

 "nonspecific" background binding usually associated with inadequately 

 differentiated slides (see below). 



2. Protein Interference and Effect of pH 



Basic dye binding by nucleic acids in tissue sections involves a complex 

 equilibrium between many different factors. It is doubtful if all nucleic 

 acid phosphoryl groups are ever active in dye binding. Some may be tied 

 to the denatured tissue proteins, since without this or other linkage the 

 lower polymers, at least, would be expected to be removed during certain 

 steps in the process of histological preparation. The extent to which the 

 dye can "compete" with protein basic groups in a fixed tissue is not clear. 

 The situation is obviously different from the in vitro competition shown 

 between dyes and proteins in solution. Protein inhibition can be made com- 

 plete in tissue sections, so that staining is abolished, by incubating slides 

 for a short time in protein solutions. '- 



Proteins, whether bound directly to nucleic acids or not, contain a large 

 number of ionizable groups which can obviously alter electrostatic fields in 

 their vicinity. The tremendous effect that protein basic groups have upon 

 nucleic acid binding may readily be seen by study of the effects of blocking 

 agents. Amino groups may be acetylated on slides by treating them with 

 100 % acetic anhydride for 1 hour at room temperature'^ or may be blocked 

 with a solution of chloramine-T.'* The amino groups may be partially re- 

 moved with nitrous acid (15% sodium nitrite in 3 parts of 50% ethanol to 

 1 part glacial acetic acid).'' After such treatments nucleic acid binding may 

 be increased several times (Fig. 1), and areas that were practically un- 

 stained before treatment may be strongly colored, as shown by Alfert" for 

 methyl green. 



The presence of protein amino groups is almost certainly responsible for 

 modifying the effect of pH on nucleic acid binding. As can be seen from 

 the data of Herrman, Nicholas, and Boricious* graphed in Fig. 2, toluidine 

 blue adsorption of PNA in solution was found to be practically constant 

 from pH 3.2 to 6.8 (curve A). With the same concentration of dye and 

 nucleic acid, the addition of fibrinogen gave the values shown by curve B, 

 and the binding of dye by muscle fibers by curve C. At pH 6.8 binding of 

 muscle fibers was greater than that of pure nucleic acid solutions presum- 

 ably because of the added effects of carboxyl binding. At lower pH the 



'2 J. B. French and E. V. Benditt, Federation Proc. 11, 415 (1952). 



3'M. Alfert, Biol. Bull. 103, 145 (1952). 



^* L. Monn6 and D. B. Slautterback, Arch. Zool. [2] 1, 455 (1951). 



