Affinity-labeled peptides obtained from the combining region of protein 460. Light chain labeling patterns using dinitrophenyl based photoaffinity labels

Abstract

Two radioactive photoaffinity reagents, based on the 2,4-dinitrophenyl group, have been used to label a homogeneous mouse myeloma protein (protein 460). After separation of the heavy and light chains, a portion of the light chains was allowed to react with maleic anhydride and then digested with trypsin while another portion was digested directly with trypsin. With one of the reagents, 2,4-[3H]dinitrophenylalanyl diazoketone, which labels the light chain predominantly, nearly all of the reagent was found attached In a previous paper (Yoshioka et al., 1973), we have reported the labeling of the combining region of protein 460 with two radioactive photoaffinity reagents based on dinitrophenyl (Dnp).* 1 *This is one of several haptens known to bind to the combining region of this protein (Jaffe et Rosenstein et al., 1972). One Dnp reagent, 2,4-[3H]dinitrophenylalanyl diazoketone ([3H]Dnp-AD), reacts predominantly with the light chain. The other photoaffinity reagent, 2,4-[3H]dinitrophenyl azide ([3H]Dnp-N3), is attached predominantly to the heavy chain. This paper deals with the isolation of light chain peptides and the location of the photoaffinity-labeled amino acid residues in the light chain of protein 460 labeled with [3H]Dnp-AD and [3H]Dnp-N3.

Combining region of protein 460, a mouse γA immunoglobulin which binds several haptens. Binding and reactivity of two types of photoaffinity labeling reagents

“…After separation of the heavy and light chains, a portion of the light chains was allowed to react with maleic anhydride and then digested with trypsin while another portion was digested directly with trypsin. With one of the reagents, 2,4-[3H]dinitrophenylalanyl diazoketone, which labels the light chain predominantly, nearly all of the reagent was found attached In a previous paper (Yoshioka et al, 1973), we have reported the labeling of the combining region of protein 460 with two radioactive photoaffinity reagents based on dinitrophenyl (Dnp). * 1 *This is one of several haptens known to bind to the combining region of this protein (Jaffe et al, 1971;Rosenstein et al, 1972).…”

“…Protein 460 was labeled at reagent concentrations of 1.04 X 10"4 or 4.8 X 10"4 m and the identity of the labeling patterns at both reagent concentrations will be shown in the accompanying paper (Hew et al, 1973).…”

“…In the case of the other reagent, 2,4-[ ]dinitrophenyl azide, which labels chiefly the heavy chain, only a small fraction (15%) of the reagent which had reacted with the protein was found on the light chain. In contrast to the labeling with the diazoketone reagent, where the reagent attacked a single residue, radioactivity from azide label was found mainly in three light chain peptides which spanned residues 29-58, 62-77, and 78-108. 460 with [3H]Dnp-AD and [3H]Dnp-N3 have been described in the accompanying paper (Yoshioka et al, 1973). The light and heavy chains were reduced with a 50-fold molar excess of dithiothreitol compared to disulfides in 0.5 m Tris-HCl-6 m guanidine-HCl (pH 8.0) buffer for 4 hr at 50°.…”

“…The scope and limitations of affinity labeling are discussed. In an accompanying paper we describe the labeling patterns of the light chains of protein 460 reacted with Dnp-N3 and Dnp-AD (Hew et al, 1973).…”

Photoaffinity labeling of the combining region of myeloma protein 460. II. Interpretation of the labeling patterns

“…Whether such residues are in fact contact amino acid residues for the haptenic portion of the label remains unestablished. A number of investigators have studied both myeloma proteins and antibody populations directed against Dnp1 with a number of different affinity reagents (Thorpe and Singer, 1969;Franek, 1971Franek, , 1973 Goetzl and Metzger, 1970a,b;Haimovich et al, 1970Haimovich et al, , 1972Hew et al, 1973;Yoshioka et al, 1973;Lifter et al, 1974). An ideal affinity reagent should react with the contact amino acid residues binding the haptenic portion of the reagent molecule.…”