CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 53 



The primary phosphoryl groups of nucleic acid have a pK of about 2/ and 

 consequently are negatively charged at pH levels above 2. Dye-nucleate 

 complexes are formed then, at least in large part, through salt linkages 

 between cation and anion. Evidence for this view can be found in the fact 

 that other cations, La+++, Th"^~^-++, protamines, histoncs, and fibrinogen, 

 compete with the dye for binding sites on the nucleic acid.^"^ Also, osmotic^ 

 and membrane potential studies^"'" demonstrate that simple cations such 

 as sodium may be adsorbed by nucleic acids in solution also through ion- 

 pair formation. Such ions would be expected to compete in some cases with 

 dyes for binding sites. For example, approximately a 20-fold decrease in 

 quinoline binding by PNA in solution was brought about by a 10-fold 

 increase in the concentration of sodium chloride or magnesium sulfate. ^'- 



There have been several studies of dye binding by nucleic acids in solu- 

 tion. Stoichiometric precipitation of dye-nucleate has been reported with 

 crystal violef '^^ and toluidine blue.^ Under certain conditions nucleic acid 

 depolymerization resulted in decreased binding of acridines,'*'^* quino- 

 lines,^^ methyl green, ethyl green, and malachite green,'*-" although methyl 

 green was bound stoichiometrically to polymerized DXA.* On the other 

 hand, nucleic acid depolymerization increased the uptake of pyronin Y'* 

 and rosaniline,'* but had no effect on the degree of binding of Victoria blue, 

 crystal violet,'* methylene blue,'^ or sodium ions.'' It has been suggested 

 that the decrease in binding with depolymerization may be associated with 

 altered steric'*-" or electrostatic'^ conditions of the nuelcic acid molecule. 

 The increase in binding shown with pyronin and rosaniline may involve 

 the removal of steric hindrance, alteration in the configuration of charge 

 centers, or the opening up of new binding sites.'* 



Other factors in addition to electrostatic charge are undoubtedly of ini- 



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 *E. G. Kelley, J. Biol. Chem. 127, 55 (1939). 



6 N. B. Kurnick and A. E. Mirsky, J. Gen. Physiol. 33, 265 (1950). 



7 A. E. Mirsky and H. Ris, J. Gen. Phijsiol. 34, 475 (1951). 



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1" J. M. Creeth and D. O. Jordan, J. Chem. Soc. 1919, 1409. 



11 J. Shack, R. J. Jenkins, and J. H. Thompsett, J. Biol. Chem. 198, 85 (1952). 



'" F. S. Parker, Science 110, 426 (1949). 



13 R. Feulgen, Z. physiol. Chem., 84, 309 (1913). 



" N. B. Kurnick, J. Gen. Physiol. 33, 243 (1950). 



•s J. L. Irwin and E. M. Irwin, Science 110, 426 (1949). 



16 J. L. Irwin and E. M. Irwin, Federation Proc. 11, 235 (1952). 



1' N. B. Kurnick, Arch. Biochem. 29, 41 (1950). 



i» L. F. Cavalieri and A. Angelos, J. Am. Chem. Soc. 72, 4686 (1950). 



"R. Vercauteren, Nature 165, 603 (1950). 



^° R. Vercauteren, Enzymologia 14, 134 (1950). 



