bridged paramagnetic /x-amido-/x-superoxo-octaamminedicobalt ( III ) cation. Diamagnetic compounds. Dr. Schaefer next turned his attention to the /x-peroxo-decaamminedicobalt ( III ) cation, which is brown and diamagnetic. (It is obtained by reduction of the paramagnetic, superoxo compound.) He found a longer O-O bond (1.47 A.) and a nonplanar CoO-O-Co group, exactly what would occur if the extra electron is paired with the odd electron in the three electron bond. The O-O bond should then be a pure single bond, and the remaining three orbitals around each oxygen should contain two unshared electron pairs and a pair that bonds to cobalt. In the Werner's red salt, the presence of a proton on the O-O bridge was suggested in 1952 by Dr. Wayne K. Wilmarth and Dr. Lawrence R. Thompson of the University of Southern California. They based this prediction on the general tendency of the reduced unit to precipitate as an acid salt. This prediction was borne out earlier this year when Dr. John Weil and Dr. Masayasu Mori at Argonne National Laboratory found that proton exchange and oxidation-reduction reactions involving the red cation were unexpectedly slow. This evidence suggested a different geometry from that in the paramagnetic compound. The detection of the O-O-H bridge by Dr. Marsh and Dr. Thewalt clears up much of the mystery that puzzled earlier investigators. Although the geometry of the bridging O-O-H group is unusual, Dr. Marsh points out that it can be explained on the basis of simple principles of bonding. The four sp 3 orbitals of the bridging oxygen atom contain three bonding electron pairs (to the two cobalt atoms and to the other oxygen atom) and an unshared pair. This results in a formal charge of + 1 on the oxygen atom, which is compensated by the ionic character of the Co-O bonds. This picture also accounts for the pyramidal (nonplanar) arrangement of bonds about the bridging oxygen atom. Dr. Marsh and Dr. Thewalt find that coordination about the cobalt atoms in both the red and green cations is approximately octahedral. All four ethylenediamine groups of the green cation have the same conformation, although one of the groups in the red cation appears to be disordered. Disorder within the crystals of the red compound is also reflected in the way the nonbridging oxygen atom points. It lies on the one side of the four-membered -Co-N-Co-O- ring in about one third of the molecules, on the other side in two thirds of the molecules.
Reversed-phase column chromatography aids analysis, purification of tRNA's Reversed-phase column (RPC) chromatographic techniques developed by chemists and engineers at Oak Ridge National Laboratory are helping scientists learn more about the role that transfer ribonucleic acids (tRNA's) play in physiological changes in a variety of biological tissues. Scientists at ORNL, which is operated by Union Carbide for the Atomic Energy Commission, have developed RPC techniques to separate and isolate a large number of amino acid-specific tRNA's found in bacterial, crustacean, and mammalian tissue. The RPC work at ORNL is part of a long-range program, sponsored jointly by AEC and the National Institutes of Health, to develop and improve methods for separating pure macromolecules. The chromatographic systems have served two functions: analysis of tRNA in various biological tissues and preparation of highly purified samples of tRNA. The RPC separations are clearing a path to further knowledge on how tRNA is altered by physiological changes such as viral infection, embryo development, and tumor growth. A number of findings relating to the RPC systems and their biological applications were disclosed at the Southeastern Regional Meeting of the American Chemical Society, in Atlanta Ga. (C&EN, Nov. 13, page 5 1 ) . An important function of tRNA (sometimes termed soluble ribonucleic acid or sRNA) molecules is to combine with specific amino acids and carry these to ribosomal particles in the cell. There the amino acids are combined into a polypeptide chain. A tRNA molecule is specific for a particular amino acid, but all tRNA's have almost identical terminal sequences with an adenine base at one end and usually a guanosine phosphate residue at the other. Multiple forms of tRNA specific for a particular amino acid such as leucine exist. These physically heterogeneous tRNA's have been named "isoaccepting tRNA's." Their separation and identification are important as they may have different coding properties specific for a single amino acid. Also, modification in the tRNA's may play an important role in mechanisms that regulate cell changes. Interest in the chemistry and biology of tRNA has led to the development of column chromatographic methods for the preparation and analytical separation of individual tRNA species from complex mixtures pres-
ent in crude tRNA (C&EN, June 5, page 4 6 ) . Recently, for example, Dr. G. M. Tener and his coworkers at the University of British Columbia, Vancouver, have developed columns packed with benzoylated diethylaminoethylcellulose to separate and purify tRNA's from brewer's yeast [Biochem., 6, 3043 (1967)]. Organic solvent. Most liquid chromatographic systems for tRNA fractionation involve aqueous solutions only. In RPC chromatography, however, the sample to be fractionated is dissolved in an organic solvent system, and the solution supported as a thin film on an inert packing in the chromatographic column. Fractionation of the sample is completed by elution with an aqueous salt solution. A. D. Kelmers and his coworkers at ORNL have developed two RPC chromatographic systems to separate Escherichia coli. In the first system, developed by Dr. G. David Novelli, Dr. Melvin P. Stulberg, and Mr. Kelmers, dimethyldilaurylammonium chloride is dissolved in isopentyl acetate and supported on acid-washed, dimethyldichlorosilane-treated diatomaceous earth. The same support is used in the second system, developed by Dr. Joseph F. Weiss and Mr. Kelmers. But the active extractant is tricaprylylmethylammonium chloride in the diluent, tetrachlorotetrafluoropropane (Freon). To develop the chromatograms, the ORNL workers use sodium chloride gradient elution with magnesium chloride and suitable buffers. They have observed differences in the chromatographic position and multiplicity of isoaccepting tRNA's with both systems. Using these columns, followed in some cases by gel filtration chromatography, Dr. Weiss and Mr. Kelmers have prepared samples of highly purified specific tRNA's, including phenylalanine, leucine, valine, and methionine tRNA's. To determine the effectiveness of the RPC systems for separating and purifying the individual tRNA's, a rapid, micro method for determining the chemical purity of tRNA has been developed by Dr. Gerald Goldstein and his coworkers. In estimating the chemical purity of a specific tRNA sample the ORNL workers compare two analyses. The first determines the amount of terminal nucleoside adenosine in the tRNA sample (all tRNA molecules terminate with adenosine). The second analysis determines the aminoacylating ability of NOV. 27, 1967 C&EN
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the tRNA for a specific amino acid. The purer the tRNA, the closer the aminoacylating ability matches the terminal adenosine assay. The RPC system using tricaprylylmethylammonium chloride in tetrachlorotetrafluoropropane is useful for analyzing multiple isoaccepting tRNA's in tissue from a mouse plasma cell tumor, according to Dr. Wen-Kuang Yang of ORNL. He and Dr. Novelli have resolved multiple isoaccepting tRNA's for 20 amino acids from mouse plasma cell tumor, MOPC-31C. Of the tRNA's specific for the 20 amino acids, only tryptophan tRNA shows a single form, they find. More heterogeneous. Compared with E. coli tRNA, mouse tumor tRNA is more heterogeneous. This means that the mouse tumor contains more multiple isoaccepting forms specific for a single amino acid than does E. coli, Dr. Yang explains. The multi plicity of isoaccepting tRNA's for one amino acid in the mouse tumor study seems to be related to the number of tRNA's published synonym codons, the ORNL workers say. Studies are now under way at ORNL to determine the biological role that these isoac cepting tRNA's play in synthesizing heterogeneous immunoglobulins of the mammalian system. In another application of RPC chro matography to mammalian tissue, Dr. B. J. Ortwerth and Dr. Novelli have studied the effect of injected ethionine (a liver carcinogen) on tRNA in rat liver. They find that the reversedphase chromatographic profiles of ra dioactive tRNA's from rats injected with ethionine- 14 C are markedly dif ferent from those obtained from ani mals injected with methionine- 14 C. Using RPC chromatography, Dr. Novelli and Dr. Larry C. Waters have found that when the virus, bacterio phage T2, infects E. coli cells, the tRNA specific for leucine is modified in eight minutes, confirming results obtained earlier by Dr. N. Sueoka and Dr. T. Kano-Sueoka of Princeton. The ORNL pair has found that the viral in fection seems to affect the chromato graphic profile of at least three spe cies of leucine tRNA. When T2 infection is allowed to continue for about two hours, the changes observed early in the infec tion persist. In addition, new leucine tRNA peaks, which can't be detected in normal E. coli tRNA, are now ob served. With the superior resolving power of RPC chromatography, Dr. Waters and Dr. Novelli can show that after T2 infections there are quantitative changes in the first three leucyl tRNA peaks, but no qualitative change in the eluted species. In addition, they have observed a marked increase in
COLLECTOR. ORNL's A. D. Kelmers (left) and H. O. Weeren adjust fraction collector used with large-scale reversedphase chromatographic columns at right. These columns permit isolation of pure specific transfer RNA's
two components of leucine tRNA which is not detected in normal cells. In further work, Dr. Waters and Dr. Novelli have found that only the first two peaks in normal E. coli leucine tRNA are aminoacylated by crude yeast synthetases. The two leucine tRNA peaks observed late in infection don't seem to be recognized by the yeast enzyme. This suggests that the new peaks are not modified forms of the first two leucine tRNA peaks. The ORNL scientists are currently studying the origin of these two peaks. RPC chromatography is also prov ing valuable in studying the changes that take place in tRNA of brine shrimp, Artemia saline, during the animal's early development. Joe Bagshaw, Dr. F. J. Finamore, and Dr. Novelli are looking for possible changes in the population of tRNA's during growth of the embryo. The ORNL scientists use RPC chromatog raphy to fractionate the tRNA's and the amino acid-linked tRNA's. So far their results with leucyl tRNA in dicate the presence in encysted blastulae of a leucine tRNA species that's absent from the larvae. ORNL's RPC chromatographic tech niques have so far netted 1 gram of highly purified phenylalanine tRNA. Portions of this sample have been given to several scientists outside ORNL for tRNA research. As sam ples of other amino acid-specific tRNA's become available, they will be distributed to research scientists through NIH's National Institute of General Medical Sciences.
