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.
The variable portion (V,) of the mouse myeloma protein MOPC-315 (a, h 2 ) was obtained from its Fv fragment and was used to immunize rabbits. Anti-VL antibodies were found to be specific to the V, region of protein 3 15 and to precipitate V,, Fv or light chain but not Fab' or intact M315. Anti-VL 315 precipitates also the light chain of MOPC-I04E (p, xl) but not the intact protein. Intact immunoglobulins (Ig) containing h chains, although they d o not precipitate with anti-VL, inhibit the binding of anti-V, t o VL in the radioimmunoassay. Radioimmunoassay inhibition studies demonstrated that VAl and Vhz cross-react extensively and that anti-VL3 15 can be used as a general anti-Vh reagent t o detect molecules containing chains in mouse serum. Analysis of sera from several mouse strains indicated that Vh-containing molecules are present in approximately 3 % of the Ig population, whereas the SJL strain has no detectable VA-bearing molecules. In some of the antisera, anti-V,3 15 slightly cross-reacted with V,-bearing molecules. Anti-VL31 5 antibodies are not anti-idiotypes since they react with myeloma proteins M315 (anti-2,4 dinitrophenyl MOPC-104E (anti-dextran) and HOPC-1 (specificity unknown) and with most s,erum Ig containing h chains. I n view of the similarity of mouse 1 chain sequences, it is suggested that most anti-VL315 is "anti-framework'' antibody.
The antibody combining site of the mouse myeloma protein 3 15 is contained in the Fv fragment, composed of the variable region of light chain (VL) and heavy chain (VH) [Hochman, J., et al. ( 1 973) Biochemistry 12, 11 301. It was also shown that the binding site for the dinitrophenyl (Dnp) ring is located in VL Biochemistry 16, 3 1541. We now describe the solid phase synthesis of a linear polypeptide of 1 15 amino acid residues with the sequence of VL of protein 31 5. The synthetic product was removed from the resin and the single disulfide bond was formed after reduction in 8 M urea. The synthetic material showed common antigenic determinants with native VL and 40% of it was adsorbed on anti-VL-Sepharose. Fractionation on Dnp-lysylSepharose showed that 24% of the synthetic VL can be adsorbed specifically on this column. Equilibrium dialysis with [3H] Dnp-lysine demonstrated that the affinity-purified syn-A n t i b o d i e s are a family of closely related proteins with diverse specificity. The sequence variability of antibodies of the same class is confined to the N-terminal 110-120 residues (VL or VH)] of the light and heavy chains. The principal variability is localized in three hypervariable regions in each V domain (Wu & Kabat, 1970) which are clustered together to form the antibody combining site (Amzel et al., 1974). The rest of the sequence in the V domains may be regarded as constructing a rigid framework whose three-dimensional structure is very similar in different antibodies, and to which are attached the hypervariable loops forming the specific site (Padlan et al., 1977). It is not yet possible to correlate precisely sequence and specificity, or to predict which combining site will be generated by a particular sequence. Also, it is not always clear what the relationship is between replacements in hypervariable regions and in framework residues. It is conceivable that synthetic homologues of antibodies will enable a systematic step-by-step replacement of amino acids in the V region and will help to elucidate the contribution of each segment in the V domain to the overall structure and to the specificity of the combining site. It is hoped that, if the synthetic methods will be found suitable for the synthesis of V domains, they can also be utilized to prepare synthetic antibodies with known specificity.The experience with the present methods of solid phase synthesis of proteins was predominantly with polypeptides of sizes no longer than 120-1 50 residues, such as RNase (Gutte & Merrifield, 1971), lysozyme (Sharp et al., 1973), or growth hormone (Li & Yamashiro, 1970). Previously we reported the From the Departments of Chemical Immunology (M.G., R.Z., and D.G.) and Biophysics (M.W.), The Weizmann Institute of Science, Rehovot, Israel. Receiced August 2, 1977. Supported in part by a grant from the Israel Commission for basic science. I Abbreviations used: V L and VH, variable portion of light and heavy chain, respectively; V,(s), synthetic VL: Fv, variable fragment of antibody composed of V L and VH; ...
Conformational flexibility of an antibody combining site composed of two identical V regions"
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