By P. Alexander, H. P. Lundgren
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Extra resources for A Laboratory Manual of Analytical Methods of Protein Chemistry. Volume 4
Another reaction might be the reduction of protein —SS— groups to —SH groups in the presence of an excess of a thiol compound (R'SH). This reaction is in fact an SH/SS interchange occurring in two stages : RSSR + R'SH ^ RSH + RSSR' (10a) RSSR' + R'SH ^ RSH + R'SSR' (10b) ESTIMATION OF THIOL AND DISULPHIDE GROUPS 25 A third type of reaction which might be investigated is the sulphitolysis of protein —SS— groups, which does not normally proceed to completion unless the products of reaction are removed: RSSR + S O r ^ RS" + RSSOJ (5) The main problems in following the progress of each of these reactions are to stop the reaction proceeding further and prevent the air oxidation of —SH groups, whilst the latter are being estimated.
The rate of destruction varies from one protein to another (Fig. 4) might be as successful for preventing cystine destruction as for preventing the destruction of 5-carboxymethylcysteine. 28 ANALYTICAL METHODS OF PROTEIN CHEMISTRY When complete amino acid analysis is not to be carried out and the hydrolysate is not therefore fractionated by column chromatography, there are two sound methods available for cystine analysis of the amino acid mixture, viz. 3). The former is the method of choice where coloured hydrolysates are to be analysed.
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