Immunoferritin binding to proteoglycan monomers. An electron microscopic study.

Abstract

Antibody to native bovine nasal cartilage proteoglycan monomer was shown by enzyme-linked immunosorbent assay to react with the purified hyaluronic acid binding region of the monomer. Antibody was digested with pepsin to produce F(ab')2 and labeled with glutaraldehyde-activated ferritin. F(ab')2 and F(ab')2-ferritin were reduced and alkylated to render them monovalent (Fab'). Antibody Fab' binding to native proteoglycan monomer was studied by electron microscopy of monomer reacted with ferritin-labeled antibody Fab' spread in a cytochrome c film. Ferritin-labeled antibody Fab' bound primarily at one end of the proteoglycan monomer. This binding was partly inhibitable by unlabeled monovalent antibody Fab', demonstrating immunospecificity. The end of the monomer with bound ferritin sometimes appeared as the thin segment, previously observed to bind to hyaluronic acid. These observations indicate that the hyaluronic acid binding region is at only one end of each proteoglycan monomer and that ferritin-labeled antibody Fab' selectively attaches to this part of native proteoglycan monomers. This methodology should be useful for future structural studies of isolated proteoglycans.