Glycogen, its chemistry and morphological appearance in the electron microscope. III. Identification of the tissue ligands involved in the glycogen contrast staining reaction with the osmium (VI)--iron(II) complex.

By application of appropriate blocking reactions (acetylation, de-amination, methylation and NaHSO3-treatment) it is demonstrated that the tissue ligands involved in the selective glycogen contrast staining reaction with the OsVI. FeII complex (known to be present in the combination K2OsO4K4Fe(CN)6) are the glycogen C2-C3 di-hydroxyl groups. Deliberate conversion of the diols into di-aldehydes and (di-)carboxyl groups by the application of specific oxidative agents followed by application of the OsVI. FeII-complex results morphologically in identical selective contrast staining of glycogen. By applying appropriate blocking reactions to such pre-oxidized aldehyde fixed glycogen, evidence is accumulated that K2OSO4 and K3Fe(CN)6 are unable to oxidize diols, whereas OSO4 and H2O2 are able to convert diols into carboxyl groups. From these results it is concluded that in the combination K2OSO4K4Fe(CN)6 the OsVI.FeII complex reacts with unchanged diols in the glycogen, whereas the OSO4 in the combination OSO4K3Fe(CN)6 can potentially create carbocyl groups in the aldehyde-fixed glycogen. The addition of urea to the two glycogen contrasting combinations (K2OSO4K4Fe(CN)6 or OSO4K3Fe(CN)6), also emphasizes that, although morphologically both combinations produce identical contrast stained glycogen, chemically the contrast staining is apparently obtained in a different way, as urea prevented the contrast formation in the glycogen by the combination K2OsO4Fe(CN)L, but not by the combination OSO4K3F e(CN)6.