14 INTRODUCTION 



These are: 



1. The study of absorption spectra can provide evidence of the presence 

 of only certain chemical groups in a particular site: this is not in itself 

 conclusive evidence of the presence of any one given chemical substance. 



2. When two groups are shown to exist in the same position in a cell, 

 it is not normally possible from studies of absorption spectrum to deter- 

 mine whether these groups occur in the same molecule or in different 

 molecules. 



3. The spectrum of a molecule may be rather markedly affected by the 

 adjacent or neighbouring molecules. Thus Caspersson states that the ab- 

 sorption spectrum of nucleic acid is altered to a significant degree by the 

 combination of nucleic acid with basic proteins. Fox and Danielli have 

 shown that the absorption maximum of astacine is shifted from the yellow 

 into the red by adsorption at an oil-water interface. R. A. Peters has shown 

 that a similar reversible change occurs with astaxanthine ; when astaxan- 

 thine is combined with certain proteins the color is blue, and when it is 

 removed from this combination the colour is red. An extreme example of 

 this type of behaviour is found with haem, the spectrum of which varies 

 in a very characteristic manner according to whether it is combined with 

 the appropriate protein which gives, for example, a haemoglobin, a 

 catalase, a peroxidase, a cytochrome, or a cytochrome oxidase. 



4. All quantitative studies on the absorption spectrum of different parts 

 of the cell need to be corrected for loss of light caused by scattering in 

 the specimen, and must be corrected, also, for the degree of orientation 

 of the absorbing molecules and their state of aggregation in the specimen. 

 Furthermore it must be demonstrated that the Beer-Lambert law is obeyed. 



The Use of Enzymes in Cytochemical Reactions 



A procedure which is rather commonly used is to allow a 

 purified enzyme preparation to act upon a specimen. For ex- 

 ample, Brachet has suggested that stains should be used to 

 identify the site of ribonucleic acid and that the presence of this 

 acid should be checked by the use of ribonuclease which should 

 remove all staining material. Such procedures involving en- 

 zymes do provide additional circumstantial evidence of the 

 existence of the components constituting their substrates in par- 

 ticular parts of cells. However, final proof can never be ob- 

 tained by this type of technique for the following reasons: 



1. It is never possible to establish that a particular enzyme is pure. It 

 is, of course, possible to show that a particular enzyme such as ribonuclease 

 is lacking in activity, say, toward certain peptide bonds or certain forms of 

 linkage of sugar molecules. But it is never feasible to show that a prepara- 

 tion is lacking in ability to split all the various types of bonds in substances 

 which might be involved in anchoring a dye. Consequently, there is always 



