DETERMINATION OF BETA-ISOMERIZED ASPARTIC-ACID AS THE CORRESPONDING ALCOHOL

The age-related formation of succinimides in proteins, through spontan eous deamidation of asparagine, and through cyclization of aspartic ac id, is thought to be followed by the hydrolysis of the succinimide rin g, yielding a mixture of ''normal'' aspartic acid sites and beta-isome rized aspartic acid sites (isoaspartic acid). The chemical reduction o f an isoaspartyl site to the corresponding amino acid alcohol, isohomo serine, has now been investigated as a general approach to measuring t he accumulation of isomerized residues in aging proteins. The methods employed were based on conditions previously found to be successful in reducing protein aspartic acid to homoserine. Borane was employed as the reducing agent, and was found to produce the expected amino acid a lcohols in reactions with model peptides. In addition, amino acid anal ysis revealed a complex pattern of unknown products of these reduction reactions, some of which were also evident when a much stronger reduc ing agent, lithium aluminum hydride, was used. The correlation of some of these side-products with the isomerization of the peptide suggests , unexpectedly, that the reactivity of reducing agents toward aspartyl residues and perhaps other sites in the peptide may be influenced by steric factors related to aspartyl isomerization. The borane reduction method was also applied to proteins. No detectable isohomoserine was formed either in ovalbumin, a model aged protein, or in human lens pro teins of advanced age, with conditions that fully reduced normal aspar tyl residues to homoserine. These tests thus indicate that the percent age of aspartic acid in the isomerized form in these proteins is below the limit of detectability (below approximately 5%). These results co mplement previous experimental results that have indicated a low bulk isoaspartyl content in most natural proteins.