Thiolated proteins

The following protocol represents a generalized method for protein thiolation using SATA. For comparison purposes, contrast the variation of this SATA modification method as outlined in Chapter 20, Section 1.1 for use in the preparation of antibody-enzyme conjugates. 

Add N-acetyl homocysteine thiolactone (Aldrich) to the bicarbonate reaction mixture to obtain a concentration representing a 10- to 20-fold excess over the amount of amines present. For protein thiolation, add the same molar excess of thiolactone reagent to the water reaction medium, and then slowly add an equivalent molar quantity of silver nitrate (AgNO j). Maintain the pH at 7.0-7.5 with periodic addition of NaOH. 

To remove the silver mercaptide formed from the facilitated protein thiolation reaction, add an excess of thiourea to convert all the silver into a soluble Ag(thiourea)2 complex and free the sulfhydryl modifications. 

Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. 

Protein thiolation is quantified by measurement of release of protein-bound thiols under reducing conditions alternatively, modification of protein isoelectric charge due to thiolation is detected (M17, T7, T8, T10). 

F5. Figueiredo-Pereira, M. E., Yakushin, S., and Cohen, G., Disruption of the intracellular sulfhydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquitination in neuronal cells. J. Biol. Chem. 273, 12703-12709 (1998). 

Biological systems overcome the inherent unreactive character of 02 by means of metalloproteins (enzymes) that activate dioxygen for selective reaction with organic substrates. For example, the cytochrome P-450 proteins (thiolated protoporphyrin IX catalytic centers) facihtate the epoxidation of alkenes, the demethylation of Al-methylamines (via formation of formaldehyde), the oxidative cleavage of a-diols to aldehydes and ketones, and the monooxygenation of aliphatic and aromatic hydrocarbons (RH) (equation 104). The methane monooxygenase proteins (MMO, dinuclear nonheme iron centers) catalyze similar oxygenation of saturated hydrocarbons (equation 105). ... 

One year later, a role of NO as mediator of protein thiolation in intact cells was highlighted by experiments demonstrating that endothelial cells respond to exogenous NO production with the transient S-thiolation of a number of as yet unidentified cellular proteins [34]. Similarly, S-nitrosocysteine was found to induce some S-glutathiolation in NIH-3T3 cells and rat hepatocytes. It is important to mention that other NO-derived species with a strong oxidative potential, such as peroxynitrite (ONOO-) are able to induce S-glutathionylation as is the case with the Ca-ATPase of sarcoplasmic reticulum which in vitro becomes inactivated [35]. 

Reduction of NAD to NADH makes the nicotinamide moiety susceptible to nitrosative (NO ) attack. It is theoretically possible to have transni-trosation from active-site RS-NO to NADH (NO attacks C5 of the nicotinamide moiety), thereby facilitating protein thiolate attack on the nucleotide (e.g., attack at C6 of the nicotinamide moiety or making nicotinamide a better leaving group i.e., attack at ribose Cl) (Stamler, 1994 S. Mohr,... 

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