218 RADIATION HIOLOGY 



tein.s of special fuiiction. Most of these may never be hifi,hly coiiceiitrated 

 ill aii>' one part of (he cell. Even if such an accinnuhitioii did occui, the 

 correlation of specific function with icadily accessihh' asjiects of the 

 chemistry of the protein molecuk! is rarely so definite! as to offer hope of 

 localizing many specific proteins by techni(|ues similar to those which have 

 just l)een tlescribed for nucleic acids. Natural color, as in hemoglobin, of 

 course, ofTers one opportunity for a microscopic approach (Thorell, 1947). 

 For the most part, however, methods of microscopic analysis of proteins 

 cannot be expected to give more than information concerning the approxi- 

 mate total amount of the protein mass, the fractionation of which on a 

 microscopic slide is possible to but a very limited extent. For example, a 

 considerable proportion of the histone of chromatin is split off readily 

 (PoUister and Ris, 1947). Nevertheless, the approximate analysis of 

 total protein is information of considerable importance to the broad fiucs- 

 tion of protein synthesis as the prime chemical achievement in growth, 

 cell division, and secretory activity (Caspersson, 1950; Pollister, 1954). 



Most methods for protein are not nearly so sensitive as are basophilia 

 and the Feulgen reaction for nucleic acids since the special reactions are 

 almost entirely those of groups at the omega ends of the amino acid resi- 

 dues, and in most proteins (protamine being one exception) no specific 

 reacting group makes up more than a small fraction of the total rmmber of 

 amino acid residvies. The reactive groups which have been used cyto- 

 logically are (a) the dibasic amino acids arginine (Serra, 1944; Thomas, 

 1946) and histidine (acidophilia, p. 219); (6) the dicarboxylic amino acid 

 glutamic acid (by alkaline basophilia, Dempsey and Singer, 1946) ; (c) 

 the sulfur-containing amino acids cystine and cysteine (see Lison, 1936; 

 Bennett, 1948) ; and (d) the aromatic amino acids tyrosine, tryptophane, 

 and phenylalanine (see p. 222). 



Specific reactions for proteins in cytological material are all adapta- 

 tions of well-known spot tests. One of the oldest of these is the Millon 

 reaction for tyrosine and tryptophane (Fig. 6-2B), which was usedbyLeit- 

 geb to identify the nature of crystals in plant cells as early as 1888. No 

 Millon test is impressive under the microscope, partly because the protein 

 cannot possibly be concentrated enough to give a strong visible reaction 

 on a slide and partly because, since everything is colored, the observer 

 does not have the benefit of the contrast to which he is accustomed in a 

 stained preparation. The test material, if present, is, of course, readily 

 detectable microscopically by objective photometric measurements. 

 The sensitivity of the Millon reaction in visible light is low (Table 6-2). 

 At the visible absorption peak (490 m/x), for a protein assumed to give a 

 Millon reaction ecjuivalent to 6.25 per cent tyrosine, the £"490 is 0.007, and 

 the protein would have to reach a concentration of over 40 per cent to give 

 a detectable extinction, 0.030, in a thickness of 1 m- (At the natural 

 ultraviolet absorption peak, 275 m^i, the absorption is no more intense.) 



