Trapping succinimides in aged polypeptides by chemical reduction

Area of Science:

• Protein chemistry
• Biochemistry
• Analytical chemistry

Background:

• Succinimide formation from aspartic acid and asparagine is a common protein aging reaction.
• The inherent instability of the succinimide ring hinders direct measurement.
• Developing methods to quantify succinimides is crucial for understanding protein degradation.

Purpose of the Study:

• To establish chemical reduction as a method for trapping and quantifying protein succinimides.
• To validate the detection of homoserine and isohomoserine as indicators of succinimide presence.
• To apply this method for analyzing trace succinimides in aging proteins.

Main Methods:

• Chemical reduction of succinimide-containing compounds using sodium borohydride.
• Analysis of reduction products (homoserine and isohomoserine) using quantitative hydrolysis and L-amino acid oxidase.
• Synthesis of isohomoserine via borane reduction of asparagine for confirmation.
• Application of the reduction method to peptide mixtures with trace succinimide content.

Main Results:

• Sodium borohydride reduction of polysuccinimide yielded homoserine and isohomoserine in amounts correlating with independent succinimide quantification.
• Isoserine identity was confirmed by resistance to L-amino acid oxidase and an independent synthesis route.
• Trace succinimides in peptide mixtures were successfully detected and quantified via their reduction products.

Conclusions:

• Chemical reduction effectively traps succinimides as stable homoserine and isohomoserine derivatives.
• This method provides a sensitive and diagnostic approach for quantifying succinimides in proteins.
• The technique is suitable for studying spontaneous aging reactions in naturally occurring proteins.