REACTIONS OF FIXATIVES WITH PROTEINS. 2 57 



anions, but some of it also transforms itself into a non-ionic, 

 sexi-covalent form, some of the six links being with the groups just 

 mentioned. ^^^ 



The industrial process results in the firm binding of chromium 

 to protein. When wool is simply steeped in the mordant at room- 

 temperature, however, this is not so.^^^ The fibre takes up the 

 mordant and becomes yellow, but the metal is loosely held and 

 can be washed out by a buffer at pH 8. 



In microtechnical fixation chromium trioxide is used at room- 

 temperature, yet the metal is firmly bound and cannot be removed 

 even by prolonged washing. ^^^ Solutions of chromium trioxide are 

 strongly acid (p. 105), and simple denaturation by acidity might be 

 thought to be partly responsible for the results; but proteins are 

 much more violently coagulated than by hydrochloric or nitric 

 acid, and are so altered that they cannot be digested by pepsin or 

 trypsin. ^^^ Less is known about the chemical changes underlying 

 the action of this fixative on proteins, in the circumstances of the 

 fixation of tissues in microtechnique, than about the changes 

 underlying the action of any other common coagulant fixative. 

 If the reaction were largely with the amino-groups of the proteins, 

 as is supposed to be the case in the mordanting of textile fibres, 

 these groups would presumably be blocked and no longer available 

 to ordinary acid dyes; yet this fixative, perhaps above all others, is 

 favourable to the action of such dyes (pp. 109 and 204). As an 

 oxidizing agent, it is likely to react with the -SH of the cysteine 

 side-group ; also with the phenyl of tyrosine, the indolyl of trypto- 

 phane, and the iminazol of histidine. It has recently been shown 

 that prolonged fixation in solutions of chromium trioxide does in 

 fact interfere with histochemical tests for the three last-named 

 amino-acids.^^-^ 



A considerable amount of misunderstanding has arisen from 

 the fact that chromium trioxide is used in the tanning of leather. It 

 has been supposed that the process throws light on fixation by 

 chromium trioxide in microtechnique. Unfortunately this is not 

 so, for anionic chromium plays no part in tanning. The chromium 

 trioxide is reduced to a basic salt, and it is cationic chromium that 

 reacts with the proteins of skin to make leather. ^^^ The reaction of 

 chromic sulphate with proteins has also been investigated chemi- 

 cally,^^ but this again is irrelevant to our subject, for the same 

 reason. Cationic chromium is seldom used deliberately as a fixa- 

 tive in microtechnique, but it may be formed by the reaction of 



