Abstract
Enzymatic fragmentation of purified human IgM obtained from a patient with macroglobulinemia was studied.
Two types of fragments were obtained by papain digestion of the subunits which had been produced by mild reduction and alkylation of IgM 1) .
One of the fragments consisted of a light chain and a part of μ-chain, and in this respect, it resembled Fab fragment of IgG. The other fragment resembled Fc fragment of IgG in that it was composed of a part of heavy chains which were not included in the Fab like fragment.
Yield of the Fab like fragment (Fab (IgMs)) was two moles per mole of the subunit of IgM.
It is proposed by Metzger et al. 2) and Small et al.3) that IgM molecule (molecular weight 900,000) is composed of five 7 S subunits (molecular weight 180,000), each of which is composed of two heavy chains and two light chains. Supporting the model proposed, they obtained two moles of Fab fragments per one mole of the subunit of IgM by trypsin digestion 4) . Yield of Fab (IgMs) in our study is also consistent with that model.
By pepsin digestion of IgM, three fragments were obtained. The three fragments were different in size, but all related structurally to the Fab portion of the IgM molecule.
The first, 5.6 S fragment (F (ab') 2 (IgM)), resembled the F (ab') 2 of IgG and was dissociated to 4.2 S fragments F (ab') by mild reduction and alkylation. Thus the 5.6 S fragment seems to be a dimer in which two monomeric 4.2 S fragments are linked by a disulfide bond.
The second, 3.3 S fragment [Fab pep (IgM)], was similar to the Fab (IgMs) obtained by papain digestion of the subunits of IgM. Since this fragment is obtained by peptic digestion without a reducing reagent, it may be concluded that Fab pep lacks a part of Fd' of F (ab') 2 on which the particular disulfide bond linking two Fab' fragments is located.
In contrast to the strong reaction between the F (ab') 2 or Fab' and anti-μ chain, the Fab pep hardly reacted with the antima chain. The fact indicated that the part of Fd' mentioned above carried some antigenic determinants of the, μ-chain which were different from Fc determinants.
The third, 2.4 S fragment [Fab piece (IgM)], consisted of a part of the i-chain and a part of the, κ-chain. This may be an important fragment because it seems to represent the aminoterminal side of the Fab (IgM) and possibly contains the antigen-binding site of the IgM.
The results of enzymatic fragmentation of human IgM are summarized and drawn sche-matically in Fig. 6 5) . In this scheme, it was assumed that the 7 S subunit of IgM is composed of two heavy chains and two light chains.
Recently, Suzuki and Deutsch reported that 8 S subunit of IgM produced by mild reduction might be composed of two heavy chains and three light chains 6) . However, we obtained no positive data indicating the presence of such additional light chains, so the model with two pairs of heavy and light chains is taken at present.
The presence of ten moles of Fab (IgMs) fragments per one mole of the IgM suggests the presence of ten antigen-binding sites per molecule of IgM antibody.
However, the number of sites of an anti-hapten antibody experimentally determined by Onoue et al. is five per molecule of native IgM or one per molecule of the subunit produced by reduction and alkylation of the IgM antibody 7).
Univalency of the subunit seems to match with the functional defect of the subunit such as incapability of agglutination, precipitation or hemolysis. This apparent discrepancy between the structure and function of the IgM has not yet been explained.
Studies of antibody activity of the component polypeptide chains, subunits or the enzymatic fragments of the IgM antibody are required to solve this problem. In this regard, the three peptic fragments obtained in our present study will be very useful in such studies.
