Bestimmung von Thiolgruppen mit AgNO3, NÄM und PCMB unter Anwendung der amperometrischen Titration

An indirect method for the determination of SH groups which is also applicable to undissolved material and proteins with sluggish reacting SH groups is described: The sample containing 0.2–0.8 ΜMol thiol is first incubated with 1.0 ΜMol of SH reagent AgNO₃, N-ethylmaleimide (NEM) or p-chloromercuribenzoate (PCMB)] and after the reaction mixed with 1.0 ΜMol of SH-glutathion. The glutathion remaining after the binding of the excessive SH reagent is finally titrated amperometrically with a 10^?3 M AgNO₃ solution at pH 7.4. This method permits a great variety both of reaction conditions and the type of the primarily used SH reagent. Comparative determinations of SH groups using AgNO₃ were carried out with various low molecular SH compounds, with muscle tissue and bovine hemoglobin. AgNO₃ is suitable for the determination of SH groups in proteins and in glutathion, but not in cysteine, cysteine ethyl ester and 3-mercaptopropionic acid, since the mercaptides of these form complexes with Ag⁺ ions. The application of NEM and PCMB is recommended generally for checking and completing the results obtained with AgNO₃.