Structural studies of human IgD: Isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation

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Abstract
A myeloma Ig[immunoglobulin]D (designated WAH) present in high concentration in plasma (.apprxeq. 3.5 g/dl) was purified in > 90% yield by a 2-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 yr without the addition of a proteolytic inhibitor, no spontaneous degradation was apparent and the isolated IgD remained structurally intact. The purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab.delta. (MW .apprxeq. 47,000) and Fc.delta. (MW .apprxeq. 80,000) fragments were generated quantitatively after 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage and CNBr fragmentation, several series of well-defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical and immunological studies, as well as for the sequence determination of the IgD .delta. chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The existence of an extra constant domain in the .delta. chain appears unlikely in view of the finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc.delta. by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the .delta.-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biolgoical role as a B [bone marrow-derived] lymphocyte receptor.

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