REACTIONS OF FIXATIVES WITH PROTEINS. 2 49 



between different parts of the same chain, or simply inaccessible 

 because the chain is so tightly folded. 



The reactive groups liberated by denaturating agents are all 

 ionizing side-chains of amino-acids. Chemists have given most of 

 their attention to those reactive groups that are particularly easily 

 shown by simple tests. It is probably for this reason that they have 

 concentrated so much on the sulphydr}d (-SH) of cysteine, the 

 disulphide (-S-S-) of cystine, the phenyl of tyrosine, and the 

 indolyl of tryptophane. It seems that the sulphydryl group, which 

 has been particularly carefully studied in relation to denaturation, 

 does not arise in this process by the splitting of disulphide bonds. 

 Its emergence from latency gives reductive properties to the protein. 



The groups that become reactive on denaturing are of great 

 importance to the histochemist, but in general microtechnique we 

 are especially concerned with the amino and carboxyl groups, for 

 these provide the main points of attachment for acid and basic 

 dyes respectively (see p. 167); and since we usually colour cyto- 

 plasm and nuclear sap with acid dyes, we are above all interested 

 in the reactivity of the amino-groups of lysine and arginine. 

 Unfortunately, not very much attention has been paid to the re- 

 lease of these particular groups from their latent condition in the 

 natural protein. This is partly due to the fact that their appearance 

 is not so dramatic, since some of them are readily accessible in the 

 protein before denaturation. 



If there were a special freeing of basic groups from a latent 

 condition, the protein would become more reactive with acids and 

 acid dyes; and conversely. Such changes would produce a shift 

 in the iso-electric point of the protein. Denaturation by alcohols 

 produces less change in the iso-electric point than fixation in 

 other ways, and that is why these substances are chosen as fixa- 

 tives when we want to study the basicity or acidity of proteins by 

 the simultaneous use of acidic and basic dye-ions (p. 262). In 

 general, there is a small shift (roughly pH 0-5) of the iso-electric 

 point in the less acid direction on denaturation. 



When a conjugated protein (lipoprotein or nucleoprotein) is 

 denatured, the protein constituent commonly separates from the 

 substance with which it was combined, and the latter then reveals 

 its presence more readily. 



The specificity of proteins, especially any immunological 

 property, is generally irreversibly lost by denaturation. Trypsin, 

 however, can be reversibly denatured: it loses and regains its 



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