Immunological Prediction of Sequence Differences among Proteins: CHEMICAL COMPARISON OF CHICKEN, QUAIL, AND PHEASANT LYSOZYMES

Abstract

Abstract Lysozyme was purified from pheasant and quail egg whites by ion exchange chromatography and gel filtration. Each enzyme was shown to be homogeneous by ultracentrifugal, electrophoretic, and immunological criteria. Quail and pheasant lysozymes were similar to chicken lysozyme in specific activity, molecular size, and extinction coefficient. The three enzymes differed from each other in electrophoretic mobility. Quantitative amino acid analysis showed that quail and chicken lysozymes were very similar in composition and that they may differ by only two amino acid interchanges. Pheasant lysozyme, however, differed from the chicken enzyme by possibly seven interchanges. The lysozymes of the three species were reduced and carboxymethylated, treated with trypsin, and subjected to peptide mapping on paper. The peptide maps of the chicken and quail lysozymes were almost indistinguishable. The peptide maps of pheasant lysozyme were, to the contrary, very different from those of the other two species. Immunological experiments with the micro-complement fixation technique were carried out with rabbit antisera directed against quail or pheasant lysozyme. Quail and chicken lysozymes were immunologically almost indistinguishable, while the pheasant enzyme was easily distinguishable from both of them. These immunological results are consistent with those described in a previous publication for antibodies directed against chicken lysozyme. The immunological relationships found among the lysozymes of these three species were unexpected. Indeed, ornithologists agree that, on the basis of nonmolecular evidence, the pheasant is more closely related to the chicken than is the quail. It is therefore especially significant that our chemical findings confirmed the earlier observation that pheasant lysozyme is immunologically less closely related to chicken lysozyme than is quail lysozyme. These data, along with those obtained by other workers on ribonuclease, phage coat protein, hemoglobin, and cytochrome c, support the hypothesis that quantitative complement fixation can be used for predicting the degree of sequence resemblance among naturally occurring proteins.