Separation of human orosomucoid major gene products using immobilized copper affinity chromatography and identification of the metal-interactive residues

Summary The major gene products of human orosomucoid, GP1 and GP2, were purified using immobilized copper affinity chromatography [Cu(II)-IMAC], with 3 mM imidazole as eluent. Both gene products bound to the Cu(II)-IMAC column in the presence of 1 M NaCl, but at different pHs. GP1 was not retained after treatment with diethylpyrocarbonate (DEPC). This modification was characterized using difference absorbance spectrophotometry and mass spectrometry. The latter provided unambiguous assignment of some of the modified residues. No correlation was observed between the modification of histidine/tyrosine and protein retention. Furthermore, removal of the carbethoxy groups of modified histidine and tyrosine 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 retnetion of GP1 involves several residues including lysines, and that a critical number of these is necessary for retention.