Thiolate proteins

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Thiolated Protein Containing Free Sulfhydryl Groups... 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

Fig. 3. Close-up view of the L-Arg binding site and surrounding protein structure. Note the sheet structure forming the roof of the active site, which is distinct from peroxidases and P450s, where hehcal structures form the distal cavity. Note, too, the H-bonds between the Cys hgand, Trp 180, and a peptide NH group. Donation of two H-bonds to the Cys ligand is a common feature found in other iron-thiolate proteins.

This enzyme [EC 1.14.15.3], also known as alkane 1-monooxygenase, lauric acid ca-hydroxylase, fatty acid hydroxylate fatty acids in the [Pg.47]

Unlike the Cd, Zn-thioneins, the respective copper-thiolate proteins were much more susceptible to oxidation. The binding energy value of the S2p core electrons of yeast copper thionein lies at 162.0 eV (Fig. 5). Ageing of protein samples or the addition of substoichiometric concentrations of H2O2 give rise to the appearence to transient oxidation states including RSSR, RSO" and sulphonic acid. The copper thiolate... 

Weser, U., Hartmann, H. J. Copper thiolate proteins (metallothionein), Copper proteins and copper enzymes, Vol. 3, p. 151, Boca Raton, CRC press 1984... 

Mansuy, D. Battioni, P. Diversity of reactions catalyzed by heme-thiolate proteins, The Porphyrin Handbook. Volume 4. Biochemistry and Binding Activation of Small Molecules Eds. Kadish, . M. Smith, . M. Guilard, R. Academic Press San Diego, 2000, pp. 1-15. 

As the focus of this review is on copper-dioxygen chemistry, we shall briefly summarize major aspects of the active site chemistry of those proteins involved in 02 processing. The active site structure and chemistry of hemocyanin (He, 02 carrier) and tyrosinase (Tyr, monooxygenase) will be emphasized, since the chemical studies described herein are most relevant to their function. The major classes of these proteins and their origins, primary functions, and leading references are provided in Table 1. Other classes of copper proteins not included here are blue electron carriers [13], copper-thiolate proteins (metallothioneines) [17], and NO reductases (e.g., nitrite [NIR] [18] or nitrous oxide [19]). 

Antibody to progesterone was also obtained by immunization with conjugates prepared from thiolated proteins. Bovine serum albumin was thiolated by reaction with 5-acetylmercaptosuccinic anhydride. After deacetylation, coupling was achieved with 6/8-bromoprogesterone. 

Fig. 11.10. Thiolation of proteins with S-acetylmercaptosuccinic anhydride (I - V). Most of the groups introduced will be acetylthio groups (III), although a few free thiol groups are also formed. The acetyl groups can easily be removed with appropriate nucleophilic reagents, yielding thiolated proteins IV. This reaction is accompanied by an undesired hydrolysis of the anhydride (V).

Elute the antibodies with suitable buffer (Section 7.1.8.2). Keep thiolated proteins under nitrogen in degassed buffer. 

Apply the mixture to a PD-10 column equilibrated with conjugation buffer to separate the thiolated protein from the low-mol-wt material (see Note 19). 

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