Folding and interaction of subunits at the antibody combining site.
The Fv fragment derived from mouse myeloma protein 315 possessing anti-dinitrophenyl (DNP) activity, is composed of two subunits, the peptide chain VL and VH. In 8 M urea there is a complete dissociation of VL and VH and an approximately twofold 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 regain of 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 x 10(-6) M) two 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. Indeed, at low protein concentration (1.5 x 10(-8 M) only the fast step is observed and renaturation is completed in less than 30s. The presence of hapten does not affect the rate of renaturation of FV. Reoxidation of FV completely reduced in 8 M urea was also found to yield a fully active Fv. Since either VL or VH have only one intrachain disulfide bond, reoxidation was performed at high protein concentration (3 mg/ml) in 8 M urea followed by dilution into neutral buffer. This demonstrates that variable domains not only exist in immunoglobulin structure but can also fold correctly independent of the rest of the peptide chains.
D000818:Animals; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004140:Dinitrophenols; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009194:Myeloma Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014508:Urea
D004140:Dinitrophenols; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D009194:Myeloma Proteins; D014508:Urea