Evidence for the absence of noncovalent bonds in the Fcmu region of IgM.
IgM isolated from the sera of five patients with Waldenström's macroglobulinemia was subjected to tryptic digestion at 60 degrees C. The Fc5mu fragments recovered from the digests were reduced by 0.05 M cysteine and alkylated by iodoacetamide, producing large quantities of an Fcmu fragment having a sedimentation velocity (see article) of 2.9S and a molecular weight of 33,500 by sedimentation equilibrium in neutral buffer. Further studies on the Fcmu fragment from one of the proteins demonstrated that it was not dissociated into smaller fragments by 5 M guanidine-HC1, even after reduction with 0.1 M 2-mercaptoethanol in 5 M guanidine at pH 7.5. The number of sulfhydryl groups released by the latter treatment indicated the presence of two intrachain disulfide bonds. These observations provide evidence that this portion of the mu-chain demonstrates minimal noncovalent interactions. Tryptic digestion of the Fcmu fragment at 37 degrees C resulted in the production of several lower molecular weight fractions. The three major fractions demonstrated apparent molecular weights of 21,000, 13,800 and 6800 by sedimentation equilibrium in 5 M guanidine-HC1. The latter fraction (fraction C) had no detectable carbohydrate and consisted of two disulfide-bonded peptides having molecular weights of approximately 3800 and 2200. Studies on the amino acid composition and amino-terminal sequences indicated that fraction C was derived from the Cmu4 homology region and consisted of residues 468 through 546 of the mu-chain with the tryptic peptides encompassing residues 492 through 514 missing.
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D007141:Immunoglobulin Fc Fragments / Q000032:analysis* / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000032:analysis*; D007075:Immunoglobulin M / Q000032:analysis*; D007146:Immunoglobulin lambda-Chains / Q000032:analysis*; D008623:Mercaptoethanol; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin; D014361:Trypsin Inhibitors; D014461:Ultracentrifugation; D014462:Ultrafiltration; D008258:Waldenstrom Macroglobulinemia / Q000276:immunology
D002241:Carbohydrates; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M; D007146:Immunoglobulin lambda-Chains; D010455:Peptides; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D008623:Mercaptoethanol; D014357:Trypsin