Structural studies of human IgD: isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation.
A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide 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 possible existence of an extra constant domain in the delta chain appears unlikely in view of our 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 biological role as a B lymphocyte receptor.
D003488:Cyanogen Bromide; D004220:Disulfides; D006801:Humans; D007072:Immunoglobulin D / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000032:analysis; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D007072:Immunoglobulin D; D007128:Immunoglobulin Fragments; D010446:Peptide Fragments; D014357:Trypsin; D010206:Papain; D003488:Cyanogen Bromide