TRIS buffer system

Preparation of gels for discontinuous SDS-containing Tris buffer systems ... 

When rat liver microsomes are incubated at 37° in TRIS buffer, there is a time-dependent loss of the activity of cholesterol 7a -hydroxylase (1). This loss of activity can be prevented if the incitoation is carried out in phosphate buffer or if fiuoride is added to the TRIS buffer system. These results are consistent with the presence of a protein phosphatase in the microsomai fraction. To determine that this activity was not due to other subcellular fractions, we prepared microsomes by our standard procedure and also by the calcium precipitation technique of Kamath et al. (2) as modified by Goodwin and Margolis (3). These preparations were tested for the presence of enzyme activities considered to be markers for other subceiiular fractions. The results of these assays... 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

The activation of H2 photoproduction by CaCl2 was observed not only with Tris buffer as an electron donor but with sucrose, dithiothreitol, or methanol as well. BaCl2, like CaCl2, enhanced the rate of H2 photoproduction by a factor of about 30. Chlorides of monovalent metals increased the reaction rate in the system Tris-Ti02-hydrogenase insignificantly [2],... 

Fig. 11. A plot of proton activity pan as a function of electromotive force (mV) for the ethylene glycol-glass electrode at +21°C and — 19°C. The response of the electrode (R ) is given by the slope of the line. The concentration of ethylene glycol is 50% by volume. The points are the experimental results in different buffer systems (a, chloroacetate b, acetate c, cacodylate d, Tris) the straight lines represent ideal behavior.

In Ref. 42 a similar approach was chosen as in Ref. 39 using stereoisomers of the type Fmoc-L-Asp-L-Asp-D-Xaa-D-Xaa (Xaa = Gly, Ala, Phe, His, Ser, Tyr). Interestingly, in part the findings are different. The ACE/MS hyphenation caused a number of practical problems affecting the reliability of the system. Surprisingly, the authors faced problems with positive ESI and were forced to use negative ionization. Because of the use of the nonvolatile Tris buffer, crystallization problems occurred frequently. Only high-EOF conditions prevented this knockout scenario. However, the description of problems and related solutions is very instructive. 

Kondo et al. simplified the buffers. They used a buffer of 25 mM Tris, 192 mM glycine for the anodic side, and 25 mM Tris, 192 mM glycine, 0.1% SDS as cathode buffer. We did not find significant differences between both buffer systems. (Kondo M, Harada H, Sunada S, Yamaguchi T (1991) Electrophoresis 12 685)... 

Synthesis of Z-Tyr-Gly-NH2 in Aqueous-DMF Solvent Media. Using this modified enzyme, we carried out the synAesis of "Z-Tyr-Gly-NH2", which has never been formed in 100% aqueous system (14), and compart with native chymotrypsin on the effect of organic solvent. To a solution of Z-Tyr-OH (315mg) in Tris buffer (pH 6.7,0.5ml), which contained N,N-dimethylformamide (DW) (0-100%), was added a solution of H-Gly-NH2 HC1 (1 Img) and native or modified chymotrypsin (2mg) in the same Tris buffer. The mixture was incubated at 20°C for 24 hours and heated at lOO C for IS minutes. The products were isolated by HPLC (ODS column, 278nm, 50% acetonitrile). Native chymotrypsin inactivated when concentration of DMF was 50%, while chemically modified chymotrypsin kept its activity even up to 80% (Table IV). 

The pH of a biological cell is controlled by the presence of natural buffers. Since protein structure is often irreversibly altered by extremes in pH, a buffer system must be maintained for protein stabilization. The importance of proper selection of a buffer system cannot be overemphasized. The criteria that must be considered in selecting a buffer have been discussed in Chapter 2. For most cell homogenates at physiological pH values, Tris and phosphate buffers are widely used. 

Alternate Protocol 3 SDS-PAGE in a Tris-Tricine Buffer System B3.1.9... 

The Tris-glycine discontinuous buffer system of Laemmli cannot be used for the separation of proteins with molecular weights < 10 to 15 kDa. For the analysis of smaller proteins, an alternative Tris-tricine buffer system is used along with an acrylamide solution that has a high percentage of cross-linker. This technique should be used when separating peptides in the size range of 1 to 20 kDa. 

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