Thiolates reactions

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. 

The following protocol describes the activation of HRP with sulfo-SMCC. Other enzymes may be activated in a similar manner. The activated enzyme possesses maleimide groups that are relatively unstable in aqueous solution. Therefore, the thiolation reaction should be coordinated with the activation process so that the final conjugation can be done immediately. Note If preactivated enzymes are obtained (Thermo Fisher), this step may be eliminated. 

The enhanced nucleophilicity in the cationic micelle is similarly expressed in the reaction with 2,4-dinitrofluorobenzene and vitamin B12 (Chaimovich et al., 1975 Nome and Fendler, 1977). In general, the extent of the rate increase observed in the thiolate reaction is somewhat smaller than that in oxyanionic reactions. This may be an important clue for elucidating the activation mechanism of the thiolate reaction. 

Dissolve SMPT (Pierce) at a concentration of 7.7 mg/ml in acetonitrile (makes a 20 mM stock solution). Alternatively, the water-soluble Sulfo-LC-SMPT may be used and dissolved at a concentration of 6 mg/ml in water (makes a 10 mM solution). This should be done just prior to adding an aliquot to the thiolation reaction. Since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester, it should be used quickly to prevent significant loss of activity. If a sufficiently large amount of protein will be modified to allow accurate weighing of Sulfo-LC-SMPT, the solid may be added directly to the reaction mixture without preparing a stock solution in water. 

The rate determining step for the disappearance of that remarkably stable (US.. SU) radical anion is the protonation of both radical anion [reaction (42)] and the free thiolate [reaction (43)] by reaction withHX... 

Acetoxycyclopropanol (8) can also be used as a source of cyclopropanone in thiolation reactions. Thus, 8 reacted with methanethiol in dichloromethane to yield l-(methylsulfanyl)cyclo-propanol (9). ... 

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