Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances: Electron stains III

Abstract

Summary Few observations giving precise information about the reactions of osmiumtetroxide with biologically important substances exist in the literature. In spite of this fact the general features of such reactions have been recognized. The most reliable information is found in the chemical literature, at least about the osmium tetroxide reactions with double bonds. These data and the results of our own study are derived from reactions under relatively well known in vitro conditions and cannot be transferred directly to a cellular system. Nevertheless such studies may give some idea of the possible reactions and help in interpreting the structures seen in ultrathin sections of osmium fixed material, where the osmium deposits are about the only “scattering” constituents. An approximate order of reactivity is suggested: Sulfhydryl groups havea direct reducing action, which may partly account for reactions with e.g. peptides, proteins and enzymes. Double bonds show about the same reactivity, an outstanding property of osmium tetroxide. Reactions with olefinic chains are exothermic and fast, but the esters formed are rather liable to hydrolyse into a diol and OsO4·2H2O. In ester formation with cyclic compounds the osmium tetroxide is firmly bound to certain double bond positions in the molecule concerned and gives rather stable products. This reaction type is of interest for fats, waxes, lecithin, the cerebrosides, vitamins, certain hormones, gallic acids and all other biological substances having a basic sterol structure. With tertiary bases the osmium tetroxide molecule easily forms stable coordination compounds, which in turn are able to accelerate the double bond reactions appreciably. Such coordination probably occurs with e.g. tryptophane. Amino groups in a terminal position and not salt linked, sulfide sulfur, hydroxyl and aldehyde groups in terminal positions and on certain carbon chain lengths, certain heterocyclic compounds and aromatic compounds with hydroxyl groups in juxtaposition are able to give intense reactions with osmium tetroxide, the latter being of importance when plant material containing tanning substances is studied. Criegee, and the workers using his method, showed that a large number of the osmium tetroxide reactions with organic substances are affected by steric conditions. This fact explains in some cases the inertness of one and the reactivity of another compound both having very similar structures. The inertness of the carbohydrates and the nucleic acids towards OsO4is one of the most striking results obtained and may be an explanation of the nonimpregnated areas in cytoplasm and nucleus in ultrathin sections of osmium fixed tissues. From empirical considerations it is suggested that protein reactions withosmium tetroxide are roughly dependent on the tryptophane, cysteine and histidine/2 content in the protein. Some enzymes obviously form exceptions to the rule. Even though a rough impression of the sites of reaction of osmium tetroxideand thus a vague interpretation of the osmium deposits in sections can be given, no conclusions can be drawn from the localisation of the osmium reactions in the heterogeneous cellular structures. The reactions of osmium tetroxide with biological material are dependent on time, temperature and concentration, and thus the results in the literature and our own studies suggest that constancy of time, temperature, object size, and composition of the fluid are essential to get comparable results when osmium tetroxide is used for fixation.