Unfolding and Renaturation of a Univalent Antihapten Antibody Fragment.

It has been shown in an earlier paper from this laboratory (1) that a univalent active fragment, obtained from rabbit antibody directed against bovine serum albumin, can spontaneously regain its original physical properties, and most of its original biological activity, after being fully unfolded. The unfolding agent used was concentrated guanidine hydrochloride. Several independent measurements indicated that all noncovalent structure of the active fragment is lost in 6 M solutions of this reagent, but that the original conformation is fully restored after dialysis to remove the reagent. The antibody fragment refolded in this way was found to have 75% of the ability of the native fragment to combine specifically with bovine serum albumin. It was concluded from these experiments that antibody specificity cannot stem from specific arrangements of noncovalent bonds, formed by interaction of unfolded polypeptide chains with antigen, as originally proposed by Pauling (2). The chemical origin of specific activity had to be ascribed instead to specific differences in amino acid sequence. Because this result is of considerable importance in attempting to establish a chemical basis for antibody specificity, it is important to confirm it by making a similar study with another type of antibody. It is the purpose of this paper to present the results of such a study, in which the antibody used was directed against the dinitrophenyl group. This antibody was first introduced by Farah, Kern, and Eisen (3) and has been used extensively by Eisen et al.1 (3, 4). Its advantage lies in the existence of a simple but sensitive method for quantitative measurement of the extent of combination between antigen and the antibody (or any active fragment). The method is based on the overlap between the absorption spectrum of DNP2 derivatives and the emission spectrum of tryptophan. This spectral overlap gives rise to an efficient transfer of energy from the tryptophan groups of the protein to the hapten, when the two are combined. This results in strong quenching of the fluorescence of the tryptophan groups. Since rabbit anti-DNP has a very strong affinity for suitable hapten molecules, such as DNP-lysine (4), the reaction between hapten and antibody (or active fragment) can be considered virtually stoichiometric. The extent of quenching can thus be used as a measure of the number of biologically active molecules which are present in a given preparation. An important feature