Amines with N-Acetylhomocysteinethiolactone

Thiolated Protein Containing Free Sulfhydryl Groups 

Thiolation of peptides and other small molecules containing amines proceeds easily with N-acetylhomocysteinethiolactone. However, protein modification often results in much lower yields unless the reaction is done for extended periods at pH 10-11. 

It has been found that silver ions catalyze the thiolation process with proteins, allowing the reaction to be completed rapidly at physiological pH (Benesch and Benesch, 1958). The addition of an equal molar concentration of AgN03 forms an insoluble complex with the thiolactone, and this in turn reacts with protein amines. 

Dissolve the amine-containing molecule to be thiolated at a concentration of 10 mg/ml in cold (4°C) 1 M sodium bicarbonate (reaction buffer). For proteins, dissolve them in deionized water at a pH of 7.0—7.5, at room temperature. Note the presence of some buffer salts, like phosphate or carbonate, are incompatible with silver nitrate. 

Add N-acetylhomocysteinethiolactone (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, then slowly add an equivalent molar quantity of silver nitrate (AgN03). Maintain the pH at 7.0—7.5 with periodic addition of NaOH. 

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