INTRODUCTION TO FIXATION 21 



protein in a latent condition are unmasked by denaturing and 

 now respond to tests for their presence. This applies to various 

 ionizing side-chains of the constituent amino-acids, especially 

 the -SH of cysteine, the -S— S- of cystine, the phenyl of tyrosine, 

 and the indolyl of tryptophane. The cause of this increase in 

 reactivity is not known, but it may be a straightening of much- 

 folded backbones and consequent exposure of previously-hidden 

 side-groups. The protein is now more accessible both to dyes and 

 to digestive enzymes. The specificity of the original protein is at 

 the same time generally lost. This is most evident if the protein is 

 or forms part of an enzyme. 



Proteins can be denatured by very diverse means. Heat alone 

 is effective, if water be present; in the complete absence of 

 moisture, however, even a temperature of 100^ C does not suffice 

 to denature. Exposure to ultraviolet light or ultrasonic waves, 

 subjection to very high pressure, and extension in extremely thin 

 films can cause the denaturing of proteins, but these methods are 

 not ordinarily used in microtechnique. 



In additive fixation a part or the whole of the fixative molecule 

 adds itself to the substance that it fixes, by making ionic or 

 covalent links. In the fixation of proteins, the link is usually with 

 the side-groups of one or more particular amino-acids. Additive 

 fixation is sometimes coagulant, sometimes not. 



An additive fixative need not necessarily alter the reactions 

 of a protein very profoundly. For instance, it might act only on 

 tyrosine side-groups, and if these were sparsely represented in a 

 particular protein, most of the amino-acids might be able to 

 retain their character and show their usual responses to reagents. 

 The rest of the protein might undergo changes similar to those 

 that occur in non-additive fixation. Thus there need not be a very 

 sharp distinction between the two kinds of fixation, so far as 

 the protein as a whole is concerned. Nevertheless, it is convenient 

 to restrict the term 'denaturing' to cases in which the protein 

 does not undergo chemical combination. 



Diff'erent primary fixatives penetrate into tissues at diff"erent 

 rates. Other things being equal, it is desirable that a fixative 

 should penetrate quickly, so that the tissue may be stabilized in 



