TRYPTIC DIGESTION OF THE SERUM IMMUNOGLOBULIN OF THE FLOUNDER, PLATICHTHYS-FLESUS

Purified serum immunoglobulin (Ig) from the flounder has been fragment ed by treatment with trypsin at 56-degrees-C. The major product of the digestion is a protein which resists further degradation over a two-h our period. This fragment constitutes approximately 40% of the Ig mole cule and on fully reducing SDS electrophoresis is composed of a single polypeptide chain of apparent molecular weight 33 kD. This is larger than the light chains and therefore indicates that the fragment is der ived only from the heavy chains of the Ig molecule. If this fragment i s composed of eight polypeptide chains, consistent with the tetrameric nature of the flounder Ig, then its molecular weight is 264 kD. Altho ugh its electrophoretic mobility on 4% non-reducing SDS gels suggests a molecular weight of 180 kD, densitometric data support the 264 kD va lue. The kinetics of the digestion indicate that the first event is th e liberation of a 50 kD fragment which is a dimer of two 25 kD polypep tides and which may be a F(ab)mu fragment. This leaves heavy chains wh ose molecular weight is reduced to 51 kD. These are subsequently furth er reduced to 33 kD in a limit digestion product which is probably the F(c)4mu fragment of the Ig molecule. The 50 kD fragment is detectable in only small quantities and is not resistant to further trypsin atta ck. After long periods of digestion the putative F(c)4mu fragment is t he only remaining high molecular weight product. The digestion product is remarkably similar in size and polypeptide composition to that obt ained after high temperature trypsinization of pentameric human IgM wh en allowance is made for the tetrameric nature of the fish Ig. However , the human F(c)5mu fragment does not have the long-term stability to continued trypsin treatment.