SEPARATION OF HUMAN OROSOMUCOID MAJOR GENE-PRODUCTS USING IMMOBILIZEDCOPPER AFFINITY-CHROMATOGRAPHY AND IDENTIFICATION OF THE METAL-INTERACTIVE RESIDUES

The major gene products of human orosomucoid, GP1 and GP2, were purifi ed using immobilized copper affinity chromatography [Cu(II)-IMAC], wit h 3 mM imidazole as eluent. Both gene products bound to the Cu(II)-IMA C column in the presence of 1 M NaCl, but at different pHs. GP1 was no t retained after treatment with diethylpyrocarbonate (DEPC). This modi fication was characterized using difference absorbance spectrophotomet ry and mass spectrometry. The latter provided unambiguous assignment o f some of the modified residues, No correlation was observed between t he modification of histidine/tyrosine and protein retention. Furthermo re, removal of the carbethoxy groups of modified histidine and tyrosin e by hydroxylamine treatment did not improve the retention. Therefore neither histidine nor tyrosine could be the critical residues in metal recognition. Results from mass spectrometric analysis of retained and unretained fractions of DEPC modified GP 1 indicated that the lysine residues 130/135 and 152 were modified significantly in both fractions , but to a relatively less extent in the retained one. We suggest that the retention of GP1 involves several residues including lysines, and that a critical number of these is necessary for retention.