Proteins preparation

Silver nitrate is astringent and a protein precipitant, which is not medically desirable. Other forms of silver have been used to avoid this problem, including coUoidal silver, silver-protein preparations, and finely divided silver metal called Katadyn silver. 

Gel filtration is very suitable for the purity check of protein preparations, especially if these have been purified by adsorptive techniques. It can be expected that high-resolution gel filtration columns will easily separate dimeric forms from monomeric forms to reveal heterogeneities of the preparations. However, a size difference of less than 20% will not result in total resolution of the peaks (although the chromatogram may be used for a qualitative judgment of the... 

White, J. W. (1992). Internal standard stable carbon isotope ratio method for determination of C-4 plant sugars in honey Collaborative trial study, and evaluation of improved protein preparation procedure. J. Assoc. Ojfic. Anal. Chem. 75,543-548. 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Umelo-Njaka, E., Nomellini, J.F., Bingle, W.H. et al. (2001) Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system. Vaccine, 19 (11-12), 1406-1415. 

Although RP-HPLC has proven its analytical usefulness, its routine application to analysis of specific protein preparations should be undertaken only after extensive validation studies. HPLC in general can have a denaturing influence on many proteins (especially larger, complex proteins). Reverse-phase systems can be particularly harsh, as interaction with the highly hydrophobic stationary phase can induce irreversible protein denaturation. Denaturation would result in the generation of artifactual peaks on the chromatogram. 

Not all studies of soy protein diets in humans have found significant reduction of plasma cholesterol. In addition to differences in diet formulations and soy protein preparations, one reason that has been proposed for these disparities is that there is a statistically significant negative correlation between the initial cholesterol level and the decrease of cholesterol achieved with the soy protein diet (r -0.48, p < 0.01) (23). In other words, those with normal or slightly elevated cholesterol levels would be expected to show less change in cholesterol as a result of the soy diet than individuals with markedly elevated lipids. Sirtori and associates (23) have followed a number of type II hyperlipoproteinemic patients who have been maintained on the soy... 

After dissociation of the 70 S ribosome into its two subunits followed by zonal centrifugation for the separation and isolation of the 30 S and 50 S subunits on a preparative scale, the ribosomal proteins were extracted by acetic acid and then separated by cellulose ion exchange chromatography and by gel filtration on Sephadex in the presence of 6 M urea. In this way all the 53 individual ribosomal proteins have been isolated (Wittmann, 1974). Proteins prepared in this manner have been used for physical studies (Brimacombe et al., 1978 Wittmann, 1982) as well as for immunological investigations (Stoffler et al., 1980 Lake,... 

As discussed above, it appears from physical studies, especially with the NMR technique, that the tertiary structure of ribosomal proteins isolated in the presence of 6 M urea and then carefully renatured under appropriate conditions is very similar to those proteins prepared in the complete absence of urea. 

As noted above, turbidity and polymer weight concentration can be directly related (Cp = or, where t is the turbidity), and the proportionality constant may be determined experimentally (cf. Zackroff et al., 1980). Microtubule protein preparations, however, usually contain a fraction of protein that does not contribute to polymer formation, and the most likely interpretation is that this fraction is composed of assembly-incompetent tubulin and nontubulin protein contaminants (Gaskin et al., 1974 Zackroff et al., 1980). Note that Eq. (30) is based on the assumption that Co represents active, polymerization-competent protomer. If only a fraction y, less than one, is active, this equation must be corrected to give... 

Setchell, K.D., Welsh, M.B., and Lim, C.K. (1987). High-performance liquid chromatographic analysis of phytoestrogens in soy protein preparations with ultraviolet, electrochemical and thermospray mass spectrometric detection, J. Chromatogr., Jan, 16, 386, 315-323. 

Evaluation of the contribution made by the proximal ligand to the oxidation-reduction equilibrium of Mb (H93 in the wild-type protein (Fig. 2)) has been more difficult because substitution of the proximal residue results in expression of apo-Mb without heme incorporation. All of the available data for these variants (Table I), therefore, derive from proteins prepared by reconstitution of purified recombinant apoprotein with exogenous heme 69, 70, 72). In those cases where trace quantities of native Mb are produced (73), heme extraction followed by reconstitution was undertaken to eliminate complications from suIfMb... 

Table IV shows the biochemical features of thomostable a-glucosidase purified 140-fold from C. thermohydrosulfuricum (Saha and Zeikus, unpublished work). The enzyme has a 162,000 molecular weight and displayed an optimum temperature for activity of 75°C. Notably, the protein preparation hydrolyzed both a-1,6 and a-1,4 linkages. This enzyme appears to play an important role in starch degradation by C. thermohydrosulfuricum because it can hydrolyze the degradation intermediates formed by the organism s unique amylopullulanase.

However, various assays utilizing nonstandardized sources of HDAC protein preparations and substrates appear to generate large variations in the resulting data. As an example. Table 6.1 summarizes the observed variations in the recently reported in vitro data for the effects of SAHA on individual H DAC isoforms [14,19-23]. HDAC isoforms used for in vitro screening assays have been expressed in Escherichia coli (HDACl, 3), Picchia, SF9 insect cells (HDACl-11) or mammalian cells (recombinant HDACl, 3) these sources may lead to differential enzyme activities. 

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