REACTIONS OF FIXATIVES WITH PROTEINS. 2 47 



light. Freezing and thawing can cause denaturation, especially of 

 lipoproteins, though freezing- drying does not: indeed, freezing- 

 drying should be used instead of fixation if undenatured protein is 

 needed. The only means of denaturing that are used in routine 

 microtechnique are heat and certain chemical agents; the former 

 is seldom used except for the fixation of blood-films. 



We cannot establish for each protein a particular temperature of 

 denaturation. Most proteins are denatured if held in water at 

 60° C for a long time, though ribonuclease is extremely resistant. 

 If a soluble protein be dried, heated to 100° C, and cooled, it 

 retains its solubility. Thus heat alone does not suffice to denature: 

 it is hot water that causes the reaction. 



Methanol, ethanol, and acetone are the chief organic substances 

 used in microtechnique as denaturing fixatives. They ally their 

 eff"ects to that of heat; or, to put it in other words, the reaction has a 

 high thermal coefficient. A very low concentration of ethanol 

 suffices to denature proteins if the temperature is above 60° C; 

 at —8° C a concentrated solution produces a flocculus, but this is 

 soluble in water at room temperature; ^^^ there is no measurable 

 denaturation of any sort below — 15° C. (It must be mentioned 

 that alcohol actually favours the solution of a few proteins, such as 

 zein and gliadin.) The alcohols and acetone dehydrate strongly, 

 but it is clear that more than this is involved in denaturation, since 

 mere drying is ineffective. 



Hydrochloric and nitric acids are denaturing fixatives, but they 

 are not very commonly used. Denaturation by heat or other means 

 occurs rather readily at the iso-electric point of each protein; at a 

 slightly more or less acid pH than this the tendency to denature is 

 less; from about pH 2-5 downwards and again above pH 10, 

 denaturation occurs at room-temperature. The strong mineral 

 acids at about o-^N are quite useful coagulant fixatives. In con- 

 centrated solution they disintegrate proteins into amino-acids. 

 Acetic acid does not ionize freely enough to produce a pH suffici- 

 ently low to coagulate proteins; in addition, the acetate ion inter- 

 feres with denaturation (p. 64). 



We must picture the proteins in the tissues of the living organism 

 as maintaining a special relation with w^ater through their various 

 water-soluble groups. This is so whether the proteins be globular 

 or fibrous, and if the latter, whether bound into gels or not. Since 

 the protein molecule is very long, the amino and carboxyl groups 

 of the terminal amino-acids are not of much significance in this 



