Immunochemical study of the β chain of Escherichia coli tryptophan synthetase and its proteolytic fragments

It has been previously reported that a limited proteolysis by trypsin of the β2 subunit of Escherichiiz coli tryptophan synthetase produces a nearly functional dimeric protein, the protomer of which consists of two large, nonoverlapping, polypeptide fragments, F1 and F2. In the study reported here, an immunochemical comparison between the native protein, the nicked protein and its two isolated proteolytic fragments was performed, leading to the following conclusions: The β2 subunit and the nicked protein, regardless of the presence or absence of the coenzyme pyridoxal phosphate and of the reduction of the Schiff base between the cofactor and the protein, are immunologically indistinguishable. This confirms the strong structural similarity between the intact and nicked protein. The failure to detect a change in the immunological reactivity of intact or nicked β2 upon removal or reduction of the coenzyme suggests that pyridoxal phosphate does not play a major role as an antigenic determinant in β2 or in modification of the conformation of the apoprotein. When the two proteolytic fragments are separated, they still cross‐react with native β2, and all the antigenic determinants of β2 are recognized either on isolated Flor on isolated F2 by antibodies directed against native β2. However, the average affinity of the antibodies for the isolated fragments is much lower than for the native or nicked protein. The results are discussed in terms of formation and arrangement of globular intermediates during the folding of the intact protein.