Comparative Tryptic Peptide Analysis Of Rabbit Heavy Chain Allotypes By Hplc

Abstract

Reversed-phase, high pressure liquid chromatography (HPLC) in a modified TFA/H₂O-TFA/acetonitrile gradient has been successfully applied to the structural analysis of serologlcally defined rabbit immunoglobulln heavy chains. This mobile phase modification yields a relatively flat baseline at high UV sensitivity. Approximately 40 distinct tryptic pep-tides were resolved from each heavy chain, representing the V_Ha⁺ (a), a2, and a3) and V_Ha⁻ (y33,30 and y33,⁻) immunoglobulin allotypes. About 30 peptides were shown to be derived from the Fc region (C_H2 and C_H3), and 8–10 peptides from the Fd region (V_H1 and C_H1). Seven Fd peptides were shared by all V_Ha⁺ and V_Ha⁻ heavy chains. The al and a2 digests each displayed one allotype-specific peptide, whereas no allotype-specific peptides were observed for the a3 heavy chain. No differences were detected between the y33,30 and y33,⁻ peptides; however, both expressed a common y-specific pep-tide. Amino acid analysis of purified al-specific and y-specific peptides Indicate that the two peptides are very similar in composition to the predicted first N-terminal tryptic peptides of V_Hal and V_Ha⁻ heavy chains, respectively.