The effect of interchain disulfide bond cleavage on the cold induced precipitation of cryoimmunoglobulins.
Selective cleavage of the interchain disulfide bonds present in the two IgG1-kappa monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0 degree C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.
D000478:Alkylation; D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D003080:Cold Temperature; D003450:Cryoglobulins / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000032:analysis; D011487:Protein Conformation; D013057:Spectrum Analysis
D003450:Cryoglobulins; D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D007075:Immunoglobulin M; D004229:Dithiothreitol