REACTIONS OF FIXATIVES WITH PROTEINS. 2 6l 



very slow below pH 3.^^^ The greatest binding of formaldehyde 

 occurs at slight alkalinity (pH 7-5 to 8).^^^ 



The importance of formaldehyde as a tanning agent for pro- 

 teins in industry has ensured a careful study of its mode of action. 

 The other important additive, non-coagulant fixative has no 

 industrial applications of this kind, since it is far too expensive for 

 practical use. This is unfortunate, for osmium tetroxide is one of 

 the most valuable fixatives in microtechnique. In electron- 

 microscopy it is pre-eminent on account of the faithfulness of its 

 fixation and the ability of chemically bound osmium atoms to 

 scatter electrons and thus make an image possible. It should be 

 noticed that osmium tetroxide could not fulfil the latter function 

 if it were a non-additive fixative. A mere deposition of unbound 

 osmium would be useless, and indeed as misleading as it often is in 

 light-microscopy. 



That osmium reacts with unsaturated lipids (olein and oleic acid) 

 was known long ago to Altmann,^ but our understanding of its re- 

 actions with other tissue-constituents has grown slowly and is still 

 very imperfect. In this chapter we are concerned only with those 

 reactions that throw light on the way in which it fixes proteins. 



The first relevant discovery was made in 1920 by Dutch chem- 

 ists,^^ who showed that osmium tetroxide could be used to convert 

 cyclohexene to cyclohexane-diol. Two points must be noticed 



Ho Ho 



Hof >,H H/ ^H.OH 



H,l I'H Hoi Jh.OH 



H2 H2 



Cyclohexene Cyclohexane-diol 



here: the reagent acts at the two ends of a double bond, and the 

 product of reaction is a compound with two adjacent hydroxyl 

 groups (a diol). 



Reactions of this sort, whether caused by oxides of manganese 



.H 



\ \ / 



CH 0<x C— 0\ 



II + ^0s02= I ;os02 



CH O^ C— Q/ 



H 



The reaction of osmium tetroxide at the ends of a double bond 



or of osmium, may proceed in two stages. First, the double bond 

 disappears and an additive compound is formed; then a second 



