[Determination of sulphydryl and disulphide groups in proteins by amperometric titration. III. Investigation of the specifity of Ag+ ions for protein SH groups (author's transl)].

The specifity of Ag+ ions for protein SH groups has been questioned frequently, even though the amperometric titration with AgNO3 is one of the most common methods for the determination of SH groups in proteins. This is due to the fact, that the formation of silver complexes in the titration of cysteine causes a consumption of AgNO3 which is too high. In order to find out if this may be true in the case of proteins, in the present work select proteins with a well known content of SH and SS groups have been titrated amperometrically in tris buffer pH 7.4 with 0.001 M AgNO3. The proteins used were hemoglobin, bovine serum albumin, ovalbumin, lysozyme, pepsin, myoglobin, and cytochrome c. The direct and the indirect titrations of (a) native, (b) denatured, and (c) NaBH4 reduced proteins showed, that the expected consumption of AgNO3 was in no case exceeded. Therefore under the conditions used AgNO3 may be considered as a specific reagent for protein SH groups. High SH values as a result of the amperometric titration of proteins with silver nitrate, which have been published occasionally, may be due to incorrect estimation of the end point of the titration. The reducibility of SS groups depends on the kind of protein. Lysozyme and pepsin were already completely reduced at 23 degrees C, whereas bovine serum albumin needed 60 degrees C. The direct titration method was useful only in some cases for the detection of all SH groups originally present in the proteins or formed by reduction with NaBH4. On the other hand the indirect titration method gave maximum values, because the slowly reacting SH groups of proteins are also allowed to react and the resulting titration curves may be evaluated correctly.