Folding and interaction of subunits at the antibody combining site

Abstract

The Fv fragment derived from mouse myeloma protein 315, possessing anti-dinitrophenyl (DNP) acitivity, is composed of 2 subunits, the peptide chains VL [L chain variable region] and VH [H chain variable region]. In 8 M urea there is a complete dissociation of VL and VH and an approximately 2-fold increase in the fluorescence emission of Fv with a characteristic red shift of 11 nm. Upon dilution of Fv from 8 M urea into neutral buffer, full activity was observed, concomitant with regain of native fluorescence spectrum. The decrease in fluorescence upon dilution from 8 M urea was used to follow the renaturation process of Fv. At relatively high protein concentration (2.5 .times. 10-6 M) 2 steps were observed during renaturation: a fast one, which is completed in less than 30 s, and a slower step, which proceeds for approximately 20 min. The fast process represents the refolding and association of VL and VH to form an active Fv, whereas the slow step is attributed to the formation of incorrect associates between VL and VH which slowly reshuffle to the thermodynamically stable active Fv. At low protein concentration (1.5 .times. 10-8 M), only the fast step is observed, and renaturation is completed in less than 30 s. The presence of hapten does not affect the rate of renaturation of Fv. Reoxidation of Fv completely reduced in 8 M urea yielded a fully active Fv. Since VL or VH have only 1 intrachain disulfide bond, reoxidation was performed at high protein concentration (3 mg/ml) in 8 M urea followed by dilution into neutral buffer. Variable domains exist in immunoglobulin structure and can fold correctly independent of the rest of the peptide chains.