DTT

The ability to perform molecular orbital (MO) calculations on metals is extremely useful because molecular mechanics methods are generally unable to treat metals. This is because metals have a wide range of valences, oxidation states, spin multiplicities, and have unusual bonding situations (e.g., dtt-pjt back bonding). In addition, the nondirectional nature of metallic bonding is less amenable to a ball and spring interpretation. 

The carbamate, prepared from the 4-nitrophenyl carbonate, is cleaved by reduction with dithiothreitol (DTT) and TEA to give the aniline, which triggers fragmentation, releasing the amine. ... 

The order Diptera (flies) contains the glow-worms Arachnocampa and Orfelia. The bioluminescence systems of dipterans do not utilize firefly luciferin in their light-emitting reactions, differing from the bioluminescence systems of coleopterans. In dipterans, it is extremely intriguing that the bioluminescence system of Arachnocampa appears different from that of Orfelia-. the former luminescence is activated by ATP, whereas the latter luminescence is stimulated by DTT but not by ATP. 

Fig. 1.15 Effect of successive additions of 10 jal of Arachnocampa luciferase (cold-water extract) to the assay mixture (90 pi) containing 2 mM ATP, 4 mM MgSC>4 and 5 il of luciferin solution (hot-water extract made with 10 mM DTT). From Viviani et al., 2002a, with permission from the American Society for Photobiology.

Dehydrocoelenterazine (D) forms an addition product (E) when treated with acetone in the presence of benzylamine (Takahashi and Isobe, 1993). Thiol compounds, such as DTT (dithiothreitol) and glutathione, also add to D, forming an equilibrium mixture containing F (Takahashi and Isobe, 1994). These adducts are chemiluminescent. 

Assay of scintillon activity. A small volume (5-50 xl) of a scintillon sample is mixed with 1ml of 50 mM Tris-HCl, pH 8.0, containing 10 mM EDTA and 1 mM DTT. To this mixture, 1 ml of 0.2 M sodium citrate, pH 5.2 (or 30 mM acetic acid) is injected and the light emission is measured. 

The solution is dialyzed against the same buffer using a hollow fiber assembly, and then added onto a column of Affi-Gel Blue (50-100 mesh, 2 x 15 cm, Bio-Rad) prepared with the same buffer. The column is washed with the same buffer. Then luciferase is eluted with 50 mM Tris-HCl, pH 8.5, containing 5mM EDTA, 3 mM DTT, and 0.5 M NaCl (Hastings and Dunlap, 1986, state that it may be preferable to omit the Affi-Gel step because of difficulties encountered). 

The eluted luciferase is precipitated with ammonium sulfate. The precipitate is dissolved in 1 mM Tris-HCl, pH 8, containing 0.1 mM EDTA, 3 mM DTT and 0.1 M NaCl, and chromatographed on a column of Sephacryl S-300 (2.6 x 97 cm) using the same buffer. Luciferase is eluted in two peaks, corresponding to the molecular weights of about 420,000 (an aggregate) and 130,000, in a ratio of about 8 1. The fractions of these two peaks are pooled separately the Mr 420,000 luciferase is concentrated by either ultrafiltration or precipitation with ammonium sulfate. 

The concentrated luciferase solution is dialyzed overnight against 4 liters of 1 mM Tris-HCl buffer, pH 8.5, containing, 0.1 mM EDTA and 3 mM DTT. Then luciferase is further purified on a column of DEAE-BioGel A (1 x 25 cm, Bio-Rad) by elution with a linear increase of NaCl from 0 to 100 mM in the same buffer as that used in dialysis. The purified luciferase had a specific activity (based on initial maximum intensity) of approximately 8.5 x 1014 quanta sec 1mD1Aj810. 

ACTH, adrenocorticotrophic hormone Met, methionine Met(O), methionine sulfoxide DTT, dithiothreitol L7, L12, Escherichia coli ribosomal proteins Met(0)-L12, L12 containing Met(O) residues a-l-PI, a-1-proteinase inhibitor Met(0)-a-l-PI, a-l-PI... 

FIGURE 5. Assay for Met(0)-peptide reductase. The assay for Met(OJ-peptide reductase is carried out in a two-step incubation. The first incubation mixture contains Met(OJ-L12, DTT and the Met(OJ-peptide reductase. At the end of this incubation, purified L12 transacetylase and [3H]acetylCoA are added and the amount of radioactivity incorporated into L7 is determined by its ability to be retained on a nitrocellulose filter. The latter represents the amount of Met(0)-L12 reduced since the oxidized protein is not a substrate for the transacetylase. 

While no other value exists for Hg (which testifies to the delicacy of the experimental approach), Farrell and McTigue80 have measured the temperature coefficient of the cpd between Hg and water. This quantity is dX/dTt from which a value of -0.4 meV K 1 has been estimated for dinterfacial structure is much more difficult than for Eaw0y which suggests that one should always proceed cautiously in trying to decode experimental quantities in molecular terms. 

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