Free thiol groups and labile disulfide bonds in the IgG fraction of human serum.

The IgG fraction was isolated from freshly taken blood serum of health persons (male and female) by column chromatography on QAE-Sephadex. After 30 min incubation with DTNB (5,5'-dithio-(2,2'-dinitro)-benzoate) the average photometrically determined quantity of thio-anions was 0.24 +/- 0.02 SH/mole IgG. Since this result remained unchanged even after 24 h incubation with DTNB, interaction with masked thiol groups cannot be assumed. If, however, the serum was incubated with DTNB for 24 h and the IgG fraction then isolated and treated with thioglycolate, an average of 1.51 +/- 0.39 moles of thio-anions were liberated per mole of IgG. This indicates that on 24 h interaction with IgG, DTNB not only reacts with free SH groups, but also opens S-S bonds by a disulfide exchange reaction. The amount of thio-anions resulting from such opened disulfide bonds was calculated as the difference between 1.51 0.24, i.e., 0.64 S-S/mole IgG. This would be accounted for if approximately 54% of the IgG fraction is composed of a subfraction containing 1 labile disulfide bone per mole. The average of 1.51 thio-anions per mole IgG resulted from 130 single values with an essentially normal statistical distribution and standard deviation well within the usual biological range.