112 AARON BENDICH 



tion constant values, and isosbestic points. These are of especial value since 

 all three may be determined on microgram quantities from the same set of 

 measurements without loss of the sample. These considerations depend 

 upon the f§;Ct (Chapter 14) that the nature and magnitude of absorption 

 of ultraviolet light by a variety of pyrimidines and purines vary wth the 

 pH of their aqueous solutions. This is attributable to ionization of one or 

 another group and is derived from the fact that the neutral and ionic species 

 of a given molecule exhibit different spectra. In order to. learn which of 

 these species (or mixture of species) is responsible for a spectrum at a 

 particular pH, the dissociation constant must be known. This has involved 

 the prior determination of (apparent) dissociation constant (s), and, from 

 this information,^"-^" spectra have been recorded at a pH so selected that 

 only the neutral, the anionic, or the cationic form (if any of these is pos- 

 sible) is present in solution. 



A variation of this method^^^'*^' is based upon the observations that the 

 curves relating pH to absorption at particular wavelengths of ultraviolet 

 light are very similar to the dissociation curves. (Such curves, for example 

 for phenol,^'^ are superimposable.) In this technique, the spectra are de- 

 termined over a wide pH range to give a -more or less continuous picture 

 of the spectral changes. This often affords a convenient method for esti- 

 mating apparent dissociation constants and the proportions of absorbing 

 forms present in solution at specific pH values. Because of the difficulty 

 (or, at times, the impossibility) inherent in potentiometric measurements 

 at very low or high pH values, a very feeble dissociation may be missed, 

 and, accordingly, an erroneous conception of ionization behavior is ob- 

 tained. Such has been the case with the second dissociation of uraciP'* 

 (cf. Shugar and Fox^^O and xanthine^'^ (cf. Cavalieri et al}^^). The second 

 dissociations are demonstrable, however, by spectrophotometry. Apparent 

 dissociation constants should be determined by both methods, and a com- 

 parison for certain pyrimidines and purines is given in Table II. It is to be 

 noted that these values are not strictly valid in the thermodynamic sense 



3'i D. J. Brown and L. N. Short, /. Chem. Soc. 1953, 331. 



3'2 J. R. Marshall and J. Walker, J. Chem. Soc. 1951, 1004. 



3'3 M. P. V. Boarland, and J. F. W. McOmie, /. Chem. Soc. 1952, 3716. 



31" P. A. Levene, L. W. Bass, and H. S. Simms, /. Biol. Chem. 70, 229 (1926). 



31B A. G. Ogston, /. Chem. Soc. 1935, 1376. 



316 A. Albert, D. J. Brown, and G. Cheeseman, /. Chem. Soc. 1951, 474. 



"T N. Whittaker, /. Chem.. Soc. 1951, 1565; 1953, 1646. 



3>8 W. Stenstrom and N. Goldsmith, /. Phys. Chem. 30, 1683 (1926). 



"' J. J. Fox and D. Shugar, Bull soc. chim. Beiges 61, 44 (1952). 



320 D. Shugar and J. J. Fox, Bull soc. chim. Beiges 61, 293 (1952). - 



321 D. Shugar and J. J. Fox, Biochim. et Biophys. Ada 9, 199 (1952). 



322 L. F. Cavalieri, J. J. Fox, A. Stone, and N. Chang, /. Am. Chem. Soc. 76, 1119 (1954). 



323 E. A. Johnson, Biochem. J. 51, 133 (1952). 



