By P. Alexander, H. P. Lundgren
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Additional resources for A Laboratory Manual of Analytical Methods of Protein Chemistry. Volume 4
Some of the results obtained by such methods in the past have been summarised by Hamilton (1960, Table 1). After these reductive treatments, the estimation of—SH groups in the reduced protein may be carried out by any of the methods described in Section IV, A. 7. It should be noted that if reactions (10a) and (10b) are driven to completion by using a large excess of thiol reagent or a dénaturant, estimation of the —SH groups produced, provides a check on the number of —SS— groups originally present in the protein.
For reactions which take more than one hour, alkyl halides are to be avoided as —SH reagents since they hydrolyse slowly. The usual approach to —SH reactivity has been to titrate the —SH protein using one of the many methods available and quote the number of "reactive" —SH groups titrated at the end point. The divergent results obtained by this approach are partly due to the use of unsuitable reagents and titration techniques, but also to the fact that there is rarely a sharp division between "reactive" and "unreactive" —SH groups.
However, current readings in this region are of no value in fixing the slopes of the two lines in the amperometric plot, so they are best avoided. Thus after recording three points in a straight line before the equivalence point, a small excess of MeHgl titrant should be added and the current read when it has stabilised (1-20 min). Further aliquots of titrant then provide the points for the excess reagent line. Constant temperature is not necessary during the course of such titrations, which normally do not take more than ESTIMATION OF THIOL AND DISULPHIDE GROUPS 35 30-45 min.
A Laboratory Manual of Analytical Methods of Protein Chemistry. Volume 4 by P. Alexander, H. P. Lundgren