Cyanogen bromide cleaves Fc fragments of pooled human IgG at both methionine and tryptophan residues.
An attempt was made to obtain fragments containing the C gamma 2 region by selectively cleaving human Fc fragments prepared from pooled IgG at Met residues using cyanogen bromide. Based on the known locations of Met residues in the Fc regions of human IgG subclasses, fragments between Met 252 and 358, comprising the C gamma 2 domains, were expected from IgGl Gm -1, IgG2, IgG3 and IgG4. Greater fragmentation of the Fc fragments occurred, however, than was predicted. Automated N-terminal sequencing identified five major points (Trp 381, 313 and 277, and Met 397 and 252) and two minor points of cleavage (Met 428 and Trp 417). The majority of cleavage points occurred at Trp rather than Met. Furthermore, cleavage at Met 358, necessary to produce C gamma 2 domains, was not detected. Control experiments verified the integrity of the Fc fragments handled in exactly the same manner without cyanogen bromide exposure and the ability of the same cyanogen bromide preparation to produce the expected cleavages at Met of sperm whale apomyoglobin without fragmentation at Trp. Cleavage at Met 358 did not occur presumably because of the difficulty associated with cyanogen bromide cleavage at Met-Thr peptide bonds. Cleavage at Trp probably occurred by way of halogen promoted oxidation of the indole nucleus with resultant peptide bond fissure. These observations show that cyanogen bromide cleavage of pooled human Fc fragments is not selective for Met, but also cleaves at Trp residues. The resultant fragmentation of the C gamma 2 region coupled with the inability to make the required cleavage at the 358-359 Met-Thr bond resulted in the inability to produce fragments comprising the C gamma 2 domains. The reasons for the selective cleavage at Met in some proteins and the cleavage at both Trp and Met in others are not known.
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D008715:Methionine; D010446:Peptide Fragments / Q000302:isolation & purification; D014364:Tryptophan
D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D010446:Peptide Fragments; D014364:Tryptophan; D008715:Methionine; D003488:Cyanogen Bromide