Fractionation methods circular

H. citelli, H. diminuta and H. microstoma, one of which is mitochondrial DNA (mtDNA) (394). The mtDNA of H. diminuta has been isolated (118) and has been shown to be a typical circular molecule. The characteristics of H. diminuta DNA are shown in Table 6.11. In contrast, E. multilocularis and E. granulosus produced two distinct DNA bands after fractionation in caesium chloride, but there was no evidence that the DNA from either band represented mtDNA (493). There is presumably so little mtDNA in comparison to nuclear DNA in these organisms that it is completely masked in preparations of total DNA by this method. That this is the case has been shown by a recent study (976), where a different procedure, based on the selective precipitation of nucleic acids by cetyltrimethylammonium bromide (CTAB), was employed to extract mtDNA from isolated mitochondria. Some 300 g and 50 g, respectively, of Taenia spp. and Echinococcus sp. tissue yielded approximately only 1 ng mtDNA. 

The first resolution of trioxalatocobaltates was accomplished in 1916 with strychnine. The method of spontaneous crystallization of antipodes from a racemic mixture of a complex was first demonstrated with K3[Co(C204)s] above 13.2°, the optical antipodes may be crystallized and mechanically separated. In practice, however, the standard technique of fractional crystallization of the strychnine diastereoisomers has been used. Partial resolution on optically active quartz has also been reported. Selective decomposition of the antipodes by circularly polarized light... 

One important experimental result was available, the quantitative measurement of the fraction of each secondary structural element by circular dichroism (CD) on purified lipid-protein complexes. This provided a constraint that allowed a careful evaluation of the secondary structure predictions derived from the various approaches, some of which were developed for water-soluble proteins and therefore of uncertain reliability for proteins in a lipid environment. The data from these analyses were combined using an integrated prediction method to arrive at a consensus secondary structure model for each protein. The integrated method involved 36 steps, with independent predictions at each step. The final model was based on an evaluation of the various predictions, with judicious intervention by the authors. As an aid to developing the appropriate weighting of all the data, they carried out the analysis for apoE-3 without reference to the available crystal structure (Wilson et al., 1991), then used the known structure of the HDL-binding amino-terminal domain of apoE-3 as feedback to reevaluate the weighting. 

Circular dichroism measurement is a convenient method to detect the conformation of molecules,especially protein molecules [1,2]. We used this method to pigment-protein complexes,PSII particles and chloroplasts of higher plants. The helix, yfl-pleated sheet, -twist,and unordered coil forms of proteins each has a characteristic circular dichroism (CD) spectrum, the CD spectrum of a protein molecule is the sum of contributions of these forms. We can determine the fractions of each form in a protein molecule from the CD spectrum of the protein. 

The methods which do not require separation are not submitted to this constraint. They include physical methods such as fluorescence emission (1,2), ultraviolet absorption (3), or circular dichroism (4), which have been used in binding studies with ATPases. However these methods are often difficult to relate the signal amplitude with the fraction of bound ligand. Another method which is theoretically suitable in every situation, particularly for low affinity systems, is equilibrium dialysis. 

Sensitive immunoassays specific for PAH-DNA adducts allow the detection of one adduct per 10 nucleotides. Fluorimetry has also been used as an alternative detection method to immunoassays. In another method, isomeric tetrols of PAH are liberated by acid hydrolysis of the DNA-PAH adducts and analyzed by LC with fluorescence detection. Structural studies and detailed characterization of PAH metabolites and their conjugates has been performed by trapping the corresponding fractions at the exit of the LC system. As an example, the total characterization of the in t /tro-formed benzo(a)pyrenetetra-hydrodiol-epoxide-guanosine adduct has been achieved by a combination of nuclear magnetic resonance, circular dichroism, and mass spectroscopic techniques. 

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