K. Randerath

An evaluation of film detection methods for weak β-emitters, particularly tritium☆

Abstract A sensitive film detection method for the visualization of weak β-emitters on chromatograms is reported. The method allows one to visualize 2–3 nanocurie 3 H/cm 2 /day (about 1 nanocurie/day for an average thin-layer spot). It is thus 25–50 times more sensitive than known procedures for film detection of tritium on chromatograms. Exposure at low temperature and in the presence of an optimum amount of seintillator is essential. The data presented for tritium are compared with data on the film detection of 14 C-labeled compounds and analyzed in terms of the reciprocity failure of photographic emulsions at low intensities of light. Our results indicate that, in the presence of scintillator, the visualization of tritium is primarily due to the interaction of photons with the emulsion (fluorography), whereas the detection of low activities of 14 C ( 2 ) is predominantly an autoradiographic process entailing the direct interaction of the 14 C β-particles with the emulsion.

Ion-exchange thin-layer chromatography: XIII. Resolution of complex nucleoside triphosphate mixtures☆

Abstract CTP, dCTP, UTP, dTTP, ATP, dATP, GTP, dGTP, and ITP can be completely separated from each other and from other mononucleotides by two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose. The chromatograms can be evaluated quantitatively and recoveries exceed 90%. Extracts from biological material can be chromatographed directly.

Base analysis of ribopolynucleotides by chemical tritium labeling: A methodological study with model nucleosides and purified tRNA species☆

Abstract 1. (1) A method for determining the major and minor base composition of ribopolynucleotides is described, which consists of the following steps: 1.1. ( a ) Enzymic digestion of the ribopolynucleotide to a mixture of nucleosides. 1.2. ( b ) Oxidation of the digest with periodate. 1.3. ( c ) Reduction with tritiated borohydride to labeled nucleoside derivatives. 1.4. ( d ) Two-dimensional thin-layer chromatography on cellulose. 1.5. ( e ) Liquid scintillation counting. 2. (2) The method is extremely sensitive: tritium-labeled digest derived from less than 1 μg of polynucleotide is required to evaluate the base composition, including most of the so-called minor or modified bases which are natural constituents of RNA. 3. (3) The method has been studied by using both ribonucleosides and tRNAs of known structure as model compounds. 4. (4) Conditions have been chosen for the overall procedure under which RNA constituents are preserved that are partially or completely destroyed under the usual conditions for alkaline hydrolysis of RNA. 5. (5) In the course of this investigation, molar absorptivities were determined for dihydrouridine, 1-methylguanosine, and N 2 , N 2 -dimethylguanosine.

Ion-exchange thin-layer chromatography: XII. Quantitative elution and microdetermination of nucleoside monophosphates, ATP, and other nucleotide coenzymes☆

Abstract A procedure is described by which small amounts of nucleotides can be eluted quantitatively from PEI-cellulose anion-exchange thin-layer chromatograms. The method comprises direct transfer of the compounds from the layer to a paper wick and subsequent elution from the paper. Spectrophotometric measurements show quantitative recoveries of 5′-CMP, 5′-AMP, 5′-UMP, 5′-GMP, DPN, UDPG, and ATP. Statistical analysis of a number of replicate determinations of 6–10 mμmole samples of these compounds indicates relative standard deviations of 0.9–4.8%. The technique can be used with slight modifications if larger amounts (up to a few hundred micrograms of individual compounds) are to be eluted. It is not restricted to anion-exchange layers, but can be used on plain cellulose and silica gel layers as well. A new one-dimensional separation method on PEI-cellulose thin layers is also described by which an alkaline hydrolyzate of RNA can be resolved into the constituent nucleoside monophosphates. This procedure combined with the quantitative elution procedure should provide a new sensitive technique for determining the base composition of RNA.

Analysis of nucleic acid derivatives at the subnanomole level: III. A tritium labeling procedure for quantitative analysis of ribose derivatives

Abstract Mixtures of ribonucleosides are quantitatively analyzed by a procedure involving the following steps: 1. (1) Oxidation with sodium metaperiodate. 2. (2) Reduction with tritiated sodium borohydride. 3. (3) Thin-layer chromatography on cellulose. 4. (4) Elution of labeled nucleoside trialcohol derivatives and quantitative evaluation by liquid scintillation counting. The method is over 1000-fold more sensitive than the conventional optical techniques. Ribopolynucleotides are analyzed by the labeling technique after appropriate enzymic digestion. In the course of this investigation the molar absorptivity ( e 260 ) of guanosine, measured in 0.02 M Tris-hydrochloride buffer, pH 7.4, was determined to be 12.5 × 10 3 M −1 cm −1 .

An investigation of the minor base composition of transfer RNA in normal human brain and malignant brain tumors.

A number of reports have described elevated levels of tRNA methylase activities in a variety of tumor tissues when compared with normal control tissues [e.g., l-41. Such elevated methylase activities may be due to elevated levels of the enzymes themselves or also to the absence of methylase inhibitors [5-71. Whether changes in methylase activity are always accompanied by parallel changes in the base composition of tumor tRNA has been studied considerably less than the properties of the enzymes. Berquist and Matthews [8] have reported elevated levels of some methylated purines in tRNA from a mammary adenocarcinoma and S 180 ascites tumor in mice. Viale and co-authors [9, lo] have reported substantially elevated levels of methylated purines as well as pyrimidines in tRNA from human brain tumors when compared with normal brain. Data published by Iwanami and Brown [ 1 I] for HeLa cell and L cell tRNA and by Baguley and Staehelin [ 121 for a transplantable rat leukemia, on the other hand, appear to be similar to minor base composition data reported for mammalian liver [ 12, 131 . Indirect evidence for altered tRNA species in tumors is provided by investigations of the chromatographic behavior of tRNA charged with radioactive amino acids [e.g. 14-161. The function of the modified bases in tRNA is

Analysis of nucleic acid derivatives at the subnanomolar level. (IV) Analysis of polyribonucleotides by conversion to tritiated nucleoside derivatives.

A previous communication of this series [ 1 ] reported on a procedure for quantitative ultramicroanalysis of ribonucleosides involving periodate oxidation, reduction of the resulting nucleoside dialdehydes with tritiated sodium borohydride, chromatographic separation, and liquid scintillation counting. As described in the present communication, this procedure can be utilized to analyze the base composition of ribooligoand ribopolynucleotides after appropriate enzymatic digestion. The entire procedure is represented in fig. 1 *.

Analysis of nucleic acid derivatives at the subnanomole level. (V) High resolution mapping of tritium labelled RNA derivatives

A previous communication in this series described an isotope derivative method for the analysis of ribopolynucleotides [ 11. The method involves enzymatic degradation of the polynucleotides to nucleosides, which are converted to 3H-labelled trialcohols by treatment with periodate and tritiated borohydride. The labelled derivatives are separated by thin-layer chromatography and assayed by scintillation counting. The method has thus far been applied only to polynucleotides containing the major bases adenine, guanme, cytosine, and uracil. We report in this communication separation procedures that make possible the application of this scheme to nucleoside mixtures obtained by digestion of nucleic acids or oligonucleotides containing minor constituents. An essential requirement of the method is the visualization of low activities of tritium on chromatograms [2].