260 G. R. WYATT 



and Markham for quantitative analysis of PNA's: pyrimidines are estimated as nu- 

 cleotides, a correction of 5% being applied to allow for degradation to nucleosides.^" 

 Abrams,*^ using isotope dilution to test comparable hydrolytic conditions, has demon- 

 strated about 7% destruction, probably deamination, of adenine and guanine. 



c. Hydrolysis to nucleotides 



As has long been known, PNA, unlike DNA, is readily hydrolyzed to 

 mononucleotides. [Cf. Chapter 11, and also Brown and Todd, Chapter 12.] 

 This may be achieved with N NaOH or A^" HCl at room temperature; the 

 latter is less convenient because the nucleic acid dissolves only slowly and 

 there is danger of splitting purine glycosidic linkages.^" Chargaff et al.^^ 

 used pH 13 to 14 at 30° overnight to degrade PNA to nucleotides prior to 

 chromatographic separation. Boulanger and MontreuiP" used 0.5 N NaOH 

 at 20-22° for 18 hours, or concentrated ammonia solution (D° = 0.925) 

 at 45° for 8 days, with good results, although ammonia under other con- 

 ditions is liable to produce nucleosides. At 37°, N alkali causes partial de- 

 amination of cytidylic acid, but 0.3 N alkali does not." This was confirmed 

 by Davidson and Smellie,^^ who used 0.3 A^ KOH at 37° for 18 hours to 

 convert PNA to nucleotides, the potassium being removed as the perchlor- 

 ate prior to separation of nucleotides by electrophoresis on filter paper. 

 Crosbie et al}^ analyzed a number of samples of PNA by this method as well 

 as by hydrolysis in N HCl and in concentrated HCIO4 in conjunction with 

 paper chromatography: alkaline hydrolysis gave consistently higher results 

 for uracil, for cytosine, and for total recovery in terms of phosphorus than 

 either of the other procedures. Alkaline hydrolysis followed by^electro- 

 phoretic separation of nucleotides appears to be the most reliable method 

 yet devised for microanalysis of PNA. [Cf. Smith, Chapter 8.] 



3. Quantitative Technique 



Small volumes of hydrolysates or other solutions can be measured on to 

 filter paper for quantitative chromatography with a variety of micropipets 

 and burets. Especially convenient are self-filling capillary pipets,^° which 

 are not difficult to make; those available commercially often have exces- 

 sively thick tips and a bore so fine that they are liable to clogging and 

 drainage error. The volume which may be placed on the paper in one ap- 

 plication depends on the scale of working: in the range of 10 to 50 )ug. of 

 each purine or pyrimidine base, to be run in one dimension for about 18 



8« R. Abrams, Arch. Biochem. 30, 44 (1951). 



" D. H. Marrian, V. L. Spicer, M. E. Balis, and G. B. Brown, J. Biol. Chem. 189, 

 533 (1951). 



88 J. N. Davidson and R. M. S. Smellie, Biochem. J. 52, 594 (1952). 



89 G. W. Crosbie, R. M. S. Smellie, and J. N. Davidson, Biochein. J. 54, 287 (1953). 

 *" P. L. Kirk, "Quantitative Ultramicroanalysis," p. 22. John Wiley and Sons, New 



York, 1950. 



