Determination of Β-isomerized aspartic acid as the corresponding alcohol

Abstract

The age-related formation of succinimides in proteins, through spontaneous deamidation of asparagine, and through cyclization of aspartic acid, is thought to be followed by the hydrolysis of the succinimide ring, yielding a mixture of "normal" aspartic acid sites and beta-isomerized aspartic acid sites (isoaspartic acid). The chemical reduction of an isoaspartyl site to the corresponding amino acid alcohol, isohomoserine, has now been investigated as a general approach to measuring the 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 alcohols in reactions with model peptides. In addition, amino acid analysis revealed a complex pattern of unknown products of these reduction reactions, some of which were also evident when a much stronger reducing 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 proteins of advanced age, with conditions that fully reduced normal aspartyl residues to homoserine. These tests thus indicate that the percentage of aspartic acid in the isomerized form in these proteins is below the limit of detectability (below approximately 5%). These results complement previous experimental results that have indicated a low bulk isoaspartyl content in most natural proteins.

Protein damage and methylation-mediated repair in the erythrocyte

“…Today's industry has the capability of producing unlimited amounts of recombinant proteins, whose intrinsic instability can be responsible for the formation ofdeamidated/isomerized side products. Interestingly, useful selective chemical methods have been developed recently by Carter and McFadden for detecting either 8-isoaspartyl residues [138] or their succinimidyl precursors [139]. However, Lisoaspartyl content in many aged proteins may be quantitatively below the detection limit of these methods [138].…”

“…Interestingly, useful selective chemical methods have been developed recently by Carter and McFadden for detecting either 8-isoaspartyl residues [138] or their succinimidyl precursors [139]. However, Lisoaspartyl content in many aged proteins may be quantitatively below the detection limit of these methods [138]. The use of PCMT type II, as a tool for characterization of deamidated/ isomerized Asx in proteins, has been proposed, and it appears to be suitable even when isoaspartyl content is low, but still sufficient to cause significant structural and functional protein alterations [22,31,130,140].…”

“…Methylation was found to occur at residues Asp2 and Asp78180, which are not part of any Ca2+-binding domain. Moreover additional methyl esterified Asx residues have been identified in the sequence region 90-105, corresponding to calcium-binding loop III, residues 93-105 and region 110-141, which includes calcium-binding loop IV (residues [129][130][131][132][133][134][135][136][137][138][139][140]. Studies carried out using calmodulin purified from bovine brain revealed the key role of calcium ions in the generation of methylatable residues.…”

Trapping succinimides in aged polypeptides by chemical reduction

“…Detection of the products of the chemical reduction of polypeptides is therefore diagnostic of succinimides, and can be successfully applied at the trace sensitivity necessary for studies of naturally aging proteins. A related study of the reduction of aspartyl and fl-aspartyl residues to, respectively, homoserine and isohomoserine, is described in the accompanying manuscript (Carter and McFadden, 1994).…”

Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

“…These decreased RTS values could be explained by formation of isoaspartate during deamidation. − Previous studies have reported that isoaspartate-containing peptides may elute earlier in reversed-phase separations than corresponding aspartate-containing peptides, , and this could have resulted in the reduced RTS values. In vitro studies also suggest that isomerized forms of deamidated peptides containing isoaspartate may be more abundant than forms containing normal aspartate. , These changes could potentially impact the automated algorithm for deamidation detection. However, in the lens samples, multiple deamidated peaks of asparagine-containing peptides were relatively rare, as can be seen in the inset to Figure .…”