A CRITIQUE OF CYTOCHEMICAL METHODS 



237 



solutions in an absorption cuvette (as described in Sect. 2) is by no means 

 a reliable procedure, although this has frequently been done. 



The first such attempt at absolute quantitation was made by Caspers- 

 son (1939) who computed the amount of DNA in 24 nuclei of living grass- 

 hopper spermatocytes, assuming that distribution was homogeneous (p. 

 235 and Fig. 6-10) and that all light loss was due to specific DNA absorp- 

 tion. No correction was made for possible protein or RNA absorption, 

 or for nonspecific light loss, but it now 

 seems evident that these first two must 

 have contributed to the total weaken- 

 ing of the light (Ris, 1947; Caspersson, 

 1950, Fig. 36). 



It will be recalled that two kinds of 

 ultraviolet absorption curves (curves 

 I and II, Fig. 6-6) have been obtained 

 for intracellular nucleoprotein. No 

 absolute computations have been made 

 from type I curves except the semi- 

 quantitative nucleic acid-protein ratio, 

 from extinctions near the nucleic acid 

 and protein peaks (best described by 

 Thorell, 1947). Caspersson, however, 

 has made an elaborate effort to com- 

 pute quantities of nucleic acid, tyrosine, 

 and tryptophane from the type II 

 curves, which were formerly supposed 

 to characterize regions of nucleic acid 

 accompanied by histone type or diam- 

 ino acid-rich protein (Fig. 6-12). The 

 method of computation has not been 

 described completely enough so that 

 readers may discover just how the val- 

 ues were reached from the measured 

 curve. It is clear, however, that the 

 curve analysis depends on the validity 

 of the assumption that the protein 

 moiety is of basic type in which the 

 presence of a large proportion of dibasic 

 amino acids, as in histones, brings about a shift of the absorption peak 

 of tyrosine (as suggested very tentatively by Stenstrom and Reinhard, 

 1925). Therefore, this particular method of curve analysis is in effect 

 invalidated by the demonstration that in histones the peak is not shifted 

 toward longer wave lengths (p. 223). 



Pollister and Ris (1947) reported computation of the amount of DNA 



2500 



3100 



2700 2900 



WAVE LENGTH. A 



Fig. 6-12. An example of analysis 

 of compound ultraviolet absorp- 

 tion curve of a cell structure con- 

 taining nucleoprotein. Curve I, 

 measured absorption; curve III, 

 nucleic acid component; curve 

 IV, tyrosine coinponent; curve V, 

 tryptophane component; curve 

 II shows the sum of the compo- 

 nents. Results: tyrosine, about 

 0.1 X 10^"' mg/ix^; tryptophane, 

 0.04 X 10 '" mg/M^; nucleic acid, 

 0.6 X 10->" mg/ix\ Light refrac- 

 tion and dilTraction are, in this 

 special case, negligible, as special 

 experiments have shown. (Redrawn 

 after Caspersson, 1940, 1950.) 



