A STRICTER INTERPRETATION OF THE FERRIC FERRICYANIDE REACTION WITH PARTICULAR REFERENCE TO THE DEMONSTRATION OF PROTEIN-BOUND SULPHYDRYL AND DI-SULPHIDE GROUPS

(1) Two distinct pigments (Prussian blue and Prussian green) are found in tissues after the ferric ferricyanide reaction; they have different chemical properties. (2) Only strong reduction causes rapid formation of Prussian blue from ferric ferricyanide solutions. Conversely Prussian green may be formed by weak reduction or possibly by absorption of traces of this pigment present in ferric ferricyanide solutions. Brown or yellow pigments in tissues may appear green in the presence of low concentrations of Prussian blue. Thus no strict interpretation of green colours can be made. (3) Sulphydryl groups, phenols, indoles, pyrroles, aromatic amines, uric acid and ascorbic acid reduced ferricyanide to Prussian blue. Certain oils, aliphatic amines, aldehydes and some inorganic reagents caused the production of Prussian green. (4) Up to 5 minutes immersion in the ferric ferricyanide solution is recommended for the demonstration of sulphydryl groups in trichloracetic-alcohol fixed tissues. (5) A technique is given for the demonstration of protein-bound disulphides, involving previous thioglycollate reduction followed by the demonstration of the formed sulphydryls by 1½ minutes immersion in the ferricyanide solution. This technique demonstrates protein containing at least 4 per cent of cystine (equivalent to 1 per cent of sulphur).