Previous work from this laboratory on the antigenic structure of myoglobin (Mb) has shown that the antigenic reactivity of peptide 1–55 resides entirely in the shorter peptide 1–31 and is located within sequence 13–23. Narrowing down this region even further was achieved here by studying chemical derivatives of peptide 1–31. Five derivatives of this peptide were prepared, characterized and their immunochemistry and conformation studied. Modification of tryptophan residues 7 and 14 with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide resulted in retention of complete antigenic reactivity. In contrast, succinylation of lysine 16, or reduction of glutamic acid 18 and aspartic acid 20 (or 27) by diborane obliterated antigenic reactivity entirely. Also, a derivative carboxymethylated at histidines 12 and 24, which has been shown to retain full antigenic reactivity, lost this reactivity completely when it was succinylated at lysine 16. Reaction of peptide 1–31 with diazonium-1H-tetrazole gave rise to a derivative modified at lysine 16, histidines 12 and 24 and tryptophan residues 7 and 14. This derivative had no ability to react with antisera to myoglobin. Since none of the derivatives exhibited any changes in conformation, these results indicated that tryptophans 7 and 14 are located outside the reactive region. On the other hand, lysine 16, glutamic acid 18 and possibly aspartic acid 20 are located within the antigenic reactive site. This information, together with our previously reported studies, has shown that the first third of the myoglobin molecule (i.e. sequence 1–55) has only one antigenic reactive region and it is located within (but does not necessarily include all of) sequence 15–23. Glycine 23 is probably unessential for its reactivity. From the present findings, and the overall strategy of approach, it was concluded that, at this level of coarse delineation (i.e. down to 8–9 residues), reactive regions are surprisingly sharp but may be more diffuse in unfolded proteins. A reactive region is invariably so but its efficiency varies with the serum. When a reactive region is segmented, its two portions may react but with decreased efficiency. Therefore, before fragmentation of a protein for immunochemical studies, it is best to confirm that the location of scission is outside a reactive region. Other precautionary steps and critical analysis of approaches employed in studying the antigenic structures of proteins are discussed.
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Apr 1, 1974
R Allan 
