Fc intermediate (Fc I), a papain-generated fragment of human IgG, intermediate in charge, molecular weight and cleavage between the Fc and Fc' fragments of IgG

Abstract

Fc intermediate (Fc i) is a papain-generated fragment of human IgG which is intermediate in charge, mol. wt and state of cleavage between the Fc and Fc' fragments of IgG. It is composed of two polypeptide chains of unequal mol. wt held together by non-covalent bonds between the Cγ3 regions. The larger polypeptide chain has both a Cγ2 and Cγ3 domain and its N-terminus is at Leu 235 (60%) and Leu 234 (40%) (IgG1 Eu numbering). The smaller polypeptide chain is composed of a Cγ3 domain with its N-terminus at Gly 341. The carboxy-termini obtained by carboxypeptidase digestion and by a computer program which determined the most probable sequences by fitting the amino acid compositions to the sequence of IgG Eu Fc were heterogeneous involving residues 440–446 for the large polypeptide chain and 429–436 for the small one. The calculated mol. wt of the large polypeptide chain was 26,183, assuming the N-terminus at Leu 234 and C-terminus at 446 and including the carbohydrate moiety. The calculated mol. wt for the small polypeptide chain was 10,682, with the N-terminus at 341 and assuming the C-terminus at 434, for a combined mol. wt of 36,865 for the fc i fragment. Sedimentation equilibrium ultracentrifugation of Fc i under non-dissociating conditions showed an M n of 36,200 ± 1200, an M W of 36,400 ± 600 and an M z of 37,000 ± 300 g/mole. The best yields of Fc were obtained with a 6-hr digestion and the best yields of fc 1 and Fc' were obtained with digestion for 18hr in phosphate buffer. Digestion in Tris buffer for 18hr gave results similar to the 18-hr digestion in phosphate buffer except the yields of Fc' were less. This fragment may be useful for exploring biological functions of human IgG Fc. In addition, some Fc fragments obtained by papain digestion of human IgG either in phosphate or Tris buffer are not covalently bonded and are probably cleaved on the carboxy-terminal side of the interchain disulfide bonds at Leu 234 or Leu 235, the N-termini for the large polypeptide chain of Fc i. This indicates that, if disulfide bonded Fc fragments are needed, gel filtration under dissociating conditions will be necessary to remove non-covalently bonded Fc.