Identification of an Antigenic Determinant of Collagen treated with Hydroxylamine

Abstract

THE treatment of collagen with hydroxylamine, which splits ester-like bonds, produces subunits carrying newly formed aspartic acid hydroxamates^1,2. Their alcohol counterparts are not yet fully identified. A series of investigations on the immunogenicity and specificity of collagen^3,4 showed that collagen treated with hydroxylamine (HC) is an antigen which induces the formation of two types of immune sera: type 1 containing only antibodies against the area of hydroxylamine treatment (HC-antibodies); type 2 containing additionally antibodies against the general collagen structure. HC therefore acts when used for immunization like a conjugated protein. Investigation of haemagglutination inhibition showed that early sera were predominantly of type 2. Hyperimmune sera, however, were mostly of type 1. Inhibition experiments proved that sera with only HC-antibodies (type 1 immune sera) can be inhibited by HC, but are not inhibited by collagen preparations such as parent gelatine. Antisera which possess both antibodies (type 2 immune sera) are inhibited extensively by HC and to a lesser degree by parent gelatine. Additionally, inhibition experiments were carried out with peptides obtained from HC, acid-soluble collagen and insoluble collagen by treatment with trypsin or colla-genase⁵. Only peptides obtained from HC inhibited HC-antibodies and these peptides when used in sufficient amounts caused a complete inhibition of potent antisera.