84 HEWSON SWIFT 



microtome. Careful observation of specimens must accompany measure- 

 ment, and specimens must be prepared with techniques that keep variables 

 at a minimum. 



VII. Ultraviolet Absorption 



Localization of the purine and pyrimidine bases of nucleic acid through 

 their ultraviolet absorption constitutes one of the most important lines of 

 cytochemical research. The techniques involved, however, are considerably 

 more complex than those required for visible light measurement, and in- 

 terpretation of the data obtained is more difficult. The quartz or reflecting 

 optical system needed for ultraviolet studies is moderately expensive and 

 often difficult to align and focus. Further, the absorption curves obtained 

 from tissue sections are composites of several components, and the absorp- 

 tion due to nucleic acids alone is often difficult to estimate. 



Typical absorption curves on different cell regions are shown in Fig. 7. 

 Where nucleic acid concentration is high (Fig. 7A,B), the 260-m^i nucleo- 

 tide peak is prominent. Where nucleotide absorption is low or absent (Fig. 

 7D) , the peak at about 280 mn of tyrosine and tryptophan in the tissue 

 proteins is apparent. Nucleic acid absorption is much stronger than that of 

 proteins; the nucleic acid extinction at 260 m;u is about 40 times higher 

 than the extinction at 280 m/z of a similar concentration of serum albumin. 

 Thus in many cases protein absorption appears merely as a small side de- 

 flection on the nucleic acid peak. From the general shape of the absorption 

 curve, however, a qualitative idea of the nucleic acid protein ratio can be 

 obtained. 



1. Factors Influencing Absorption Curves 



Both nucleic acid and protein curves may change shape under certain 

 conditions. Nucleotide absorption changes only slightly with pH^^^ although 

 the long wavelength side of the PNA curve shows an increase in absorption 

 with increasing acidity. '^'^ Depolymerization of PNA causes a decrease of 

 about 20% at 295 m/x,'*- while depolymerization of DNA results in an in- 

 crease at 260 m/i of about 30%.'^^ Combination of DNA or PNA with some 

 metals ions, such as zinc, causes a shift in the absorption peak to higher 

 wavelengths. Treatment with perchloric acid increases PNA and DNA ab- 

 sorption, and shifts the DNA peak to 268 m^.'*' Also, the medium in which 

 nucleic acid is dispersed may affect its absorption; the peak for pure aque- 

 ous solutions of nucleic acid shifts to longer wavelengths when glycerin 

 is added. A reduction in nucleic acid absorption may occur with prolonged 



'" E. R. Holiday, Biochem. J. 24, 619 (1930). 

 '82 M. Kunitz, J. Biol. Chem. 164, 563 (1946). 

 183 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 



