Measurement of Asp/Asn Damage in Aging Proteins, Chemical Interconversion of Aspartyl Isomers, ¹⁸O Tagging of Enzymatically Repaired Aspartyl Sites, and Enzyme Automethylation at Sites of Asp/Asn Damage

Abstract

Asp/Asn damage in aging proteins, resulting from the propensity of L-Asn and L-Asp residues to spontaneously convert to a mixture of a-epimerized and 13-isomerized aspartyl products via succinimide intermediates (Figure 1), is a practical problem from the standpoint of researchers seeking to isolate and study pure proteins. In particular, the advent of protein overexpression systems and the convenience of working with large quantities of protein has made it increasingly common for investigators with no prior intention of studying spontaneous protein damage to find that a protein of interest has undergone a transformation that is ultimately found to be due to Asp/Asn damage. The first indication of this problem is generally the detection of isoforms of a polypeptide with altered chromatographic or electrophoretic properties, often as a function of a heat-step involved in the purification of the protein. Other times the formation of these spontaneously formed isoforms has been traced to a prolonged fermentor run or a lengthy storage period during the production of the protein. Though the isoforms of the protein may make up only a few percent of the total material, their presence is troubling since the purity of the protein is compromised.