Isotopically enriched media

Figure 1. EPR spectra of the molybdenum-iron protein of Azotobacter vine-landii, enriched as indicated by isolation of the protein from bacteria grown on isotopically enriched media.

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

Sample preparation. Sample concentrations in the range 0.5-2 mM (several mg cm ) are usually required, and samples must be enriched in other NMR nuclei C, IST). This can be achieved by recombinant DNA methods in isotopically enriched media. 

Fe form of the rubredoxin is produced. This medium also has an advantage in that E. coli can be grown on isotopically enriched media containing N-, S-, or Relabeled compounds so that the isotopically enriched rubredoxins can be obtained. 

We hydrolyzed ATP and ADP in 1 N and 0.1 N HC1 and in buffered solutions at pH 4j nd 8 in which the hydrolysis medium was variously enriched in °0 to either 10% or 20%. To assess the isotopic enrichment of each such solution for use in the nucleotide hydrolysis experiments, we hydrolyzed PCI, in the solution, esterified the resultant phosphoric acid/inorganic phosphate (P.) by reaction with diazomethane, and determined the isotopic distribution of the trimethyl phosphate (TMPO) by mass spectrometry. The 1 N and 0.1 N HC1 hydrolyses were allowed to proceed for 45 min and 10 hr, respectively, at 70, insuring complete conversion of ATP into AMP + 2P. The pH 8 hydrolyses were allowed to proceed for 36 hr at 70 to a point (20-25% completion) at which the ratio of ADP to AMP established that 96% and 4%, respectively, of the P. released had arisen by the primary and secondary hydrolysis steps, namely, ATP ADP + P. and ADP " AMP + P. 0The pH 4 hydrolyses were allowed to proceed for 24 hr, also at 70, to 40% completion. 

The nitrogen HMQC experiment provides information about the connectivity of nitrogens and their attached protons. For proteins, the use of HMQC normally requires isotopic enrichment of N, which is obtained by growing an organism in a medium containing a sin-... 

The interpretation of one- and two-dimensional spectra of large biomolecules such as proteins and nucleic acids is usually impossible due to the large number of highly degenerated peaks. Hence, even for the medium-sized molecules, it is necessary to use isotopic enrichment with and nuclei, and to perform triple-resonance 3D NMR experiments for resonance assignment and extraction of structural constraints. However, as we pointed out above, the resolution of conventionally acquired 3D spectra, is limited by sampling requirements. Therefore, it is rarely possible to obtain line widths close to the natural ones in a reasonable time, even for very fast-relaxing molecules. The conventional 4D spectra, such as or... 

Preparative centrifugal separations are often classified according to the phases of the media and the material to be purified, e.g., gas-gas, liquid-liquid, or liquid-solid. Gas-phase separations are very important in certain applications, e.g., uranium-isotope enrichment, but are highly specialized and not widely used. Liquid-liquid or even liquid-liquid-solid separations, on the other hand, are much more common. However, the majority of preparative separations involve the sedimentation of solid particles in a liquid medium. 

For these reasons, criticality safety quality assurance (QA) needs the qualification of the codes. The problem is complicated by the need to make these qualifications in the many situations encountered in the diverse plants, facilities, and laboratories without omitting the transport packages uranium with various enrichment, plutonium with different isotopic cbnqmsition, mediums where thermal- or epithermal- or fast-neutron population is preponderant, homogeneous or heterogeneous mediums coupled with various structure materials. 

The products of nucleosynthesis are returned to the interstellar medium by supernovae and by mass loss from red giants and novae. The processed and expelled material, isotopically enriched to varying degrees, is then incorporated in later generations of stars, where further nuclear processing takes place. Thus the Solar System was formed from material that has been processed by a number of nuclear processes in a variety of stellar environments over the lifetime of the galaxy. 

Boron trifluoride is also employed in nuclear technology by uti1i2ing several nuclear characteristics of the boron atom. Of the two isotopes, B and B, only B has a significant absorption cross section for thermal neutrons. It is used in " BF as a neutron-absorbing medium in proportional neutron counters and for controlling nuclear reactors (qv). Some of the complexes of trifluoroborane have been used for the separation of the boron isotopes and the enrichment of B as (84). 

Johnson et al. (1999) measured 5 " Se in volatilized Se, presumably in the form of alkylselenides, that was generated by cyanobacterial mats and incubated soils. For the cyanobacteria, an early sample of a vigorously growing culture yielded no measurable difference between the volatilized Se and the growth medium, whereas a later sample was enriched in the hghter isotope by 1.1 %o. In the soil volatilization experiments, four samples from two different incubated soils were analyzed. All of the samples 8 Se values were within 0.6%o of the total Se in the soil. Both the cyanobacteria and soil experiments were not highly controlled, but they do suggest that isotopic fractionation related to volatilization is small. 

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