Electron Stains

Abstract

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO++/P mole ratio of about ~ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about 1/~. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.

Electron Microscopy of Dna Molecules "Stained" With Heavy Metal Salts

“…Similar results have been obtained independently by Beer (2, 3) using uranyl nitrate at pH 3.5. The contrast in his pictures apparently is lower, and from his chemical studies (Zobel and Beer,28,29) it can be assumed that the UO2:P ratio in his preparation is ~1:2, whereas according to the results of Huxley and Zubay (14), in our preparation, the ratio should be 1 : 1 or higher, if not too much of it is lost during the washing of the grids. Even though BIBLIOGRAPHY…”

“…It has been shown that histone can take up at least one-fifth and serum albumin at least one-seventh of the amount of uranyl which DNA will take up under the same conditions (14,28). We assume that most of the "background staining" in our preparations can be attributed to this binding of heavy metals mainly by the carboxyl groups in the protein film.…”

“…For uranyl salts the subject is discussed in several recent publications (12,14,28,29). It appears that in solution at pH 3.5 a very stable complex between uranyl ion and DNA is formed with a mole ratio U O 2 : P = 1:2.…”

Phylogenetic considerations of neurosecretory granule contents: Role of nucleotides and basic hormone/transmitter packaging mechanisms.

“…We have recently identified uranaffin-positive granules in the "stem cells" of the primitive nervous system of Hydra, indicating, perhaps, an even older existence. Since the basis of the staining of the uranaffin reaction is the binding of uranyl ions to the phosphate groups of nucleotides (ZOBEL and BEER, 1961), it is possible that the dense cores identified in coelenterate neurosecretory granules using this technique, could alternatively be large concentrations of polyphosphates. Biochemical assays or ultrastructural hydrolytic digestion experiments will be necessary to positively identify the stained component as ATP.…”

“…Ultrastructural cytochemical data from our laboratory indicated that the neurosecretory granules found in the neuroendocrine "stem cells" (WESTFALL,1973) of Hydra (PAYNE and CROMEY, 1987) also contain nucleotides (or polyphosphates) based on the chemical specificity underlying the uranaffin reaction (ZOBEL and BEER, 1961). These cytochemical studies indicate that nucleotides were perhaps present in the neurosecretory granules of the neurons of the early coelenterates, which phylogenetically represent the simplest of nervous systems (i. e., nerve net).…”