The Cleavage of Rabbit Immunoglobulin G by Trypsin after Mild Reduction and Aminoethylation

It has been suggested by Noelken, Nelson, Buckley & Tanford (1965) that in rabbit IgG* the three fragments (two Fab fragments and one Fc fragment) are arranged as three compact globular entities joined by flexible regions of the heavy chains.Papain digestion (Porter, 1959) splits the molecule into these three fragments, whereas limited cleavage with pepsin (Nisonoff, Wissler, Lipman & Woernley, 1960) or CNBr (Cahnmann, Arnon & Sela, 1965) yields, after reduction, fragments that are similar to Fab fragment.Noelken et al. (1965) also suggested that the flexible region includes the disulphide bonds, between the two heavy chains, that could be reduced under very mild conditions (Palmer & Nisonoff, 1964).I now report the cleavage of rabbit IgG into three fragments by hydrolysis with trypsin; presumably cleavage takes place near one of the disulphide bonds in the flexible region.This was achieved after the conversion of the half-cystine residues into aminoethylcysteine residues by mild reduction and aminoethylation.Rabbit IgG (15mg./ml. in 0-15M-NaCI-0*2M- tris hydrochloride buffer, pH8-2) was reduced with various concentrations of dithiothreitol for 50min.* The nomenclature for immunoglobulin (IgG) and its fragments is that recommended by the World Health Organization (1964).