Controlled Deamidation of Peptides and Proteins: An Experimental Hazard and a Possible Biological Timer

Abstract

Abstract. Experiments on model peptides show that the rate of deamidation of asparaginyl residues depends strongly on the nature of neighboring residues. The natural distribution of glutaminyl and asparaginyl residues is ordered with respect to the biological lifetime of the peptides and the functional groups of the residues neighboring to glutaminyl and asparaginyl residues. The rates of deamidation of such amide peptides under physiological conditions could serve as useful timers of development and aging.The instability of the amino acids glutamine and asparagine with respect to hydrolytic deamidation of their functional side chains under relatively mild chemical conditions is well known. [1][2][3][4] The instability of glutaminyl and asparaginyl residues is a hazard during purification and primary structure determinations of peptides and proteins. In addition, now that Merrifield's solidphase peptide synthesis5-726 procedure has made the synthesis of peptides easily available to all laboratories, it seems likely that the differential stabilities of amide side chains during synthetic procedures will provide an additional problem.The experiments of T. Flatmark8-10'27 on the properties of the deamidated forms of horse heart cytochrome c show that variations occur in the rate of deamidation of different amides in the same protein molecule and that the deamidated cytochromes exhibit altered biological activity and structure. Deamidation has also been observed in numerous other proteins.11Why are these potentially unstable residues so widely distributed in nature? We suggest that controlled deamidation of glutaminyl and asparaginyl residues is a mechanism by which molecular and organismic development and aging are controlled.The rates of deamidation of glutaminyl and asparaginyl residues in proteins are probably determined by the primary and tertiary structures that govern distribution of amino acid residues surrounding the glutaminyl and asparaginyl residues in proteins. We have undertaken a study of the primary sequence dependence of deamidation of glutaminyl and asparaginyl residues in l)eptides.By comparing our results with the rates of deamidation of peptides of natural sequences and their corresponding proteins, we should also be able to make estimates of the tertiary structure dependence of deamidation. The results of our MAierrifield syntheses of the needed model peptides should aid in the prediction of difficulties during peptide synthesis and purification.

Multiple Forms of Human Glutathione S‐Transferase and Their Affinity for Bilirubin

“…Nor are the enzymes subject to hydrolysis by neuraminidase or alkaline phosphatase. We suspect that the isolated charge isomers are the result of a deamidation in vivo, a form of enzyme aging as suggested by Robinson et al [21], and one which has been clearly documented for cytochrome c [22] and rabbit muscle aldolase [23,24]. Unfortunately, the limited amount of the human enzymes that is available precludes meaningful measurements of the amide nitrogen content of these species.…”

Amino‐Acid Sequence of lac Repressor from Escherichia coli

“…Robinson etal. [47] have indicated a correlation between the number of amide residues in proteins and its biological half-life. The lac repressor monomer, with an amide content of 11 x, would be expected to have a very short half-life of 1 -10 days, in agreement with the finding that pure lac repressor is not stable with respect to its operator binding activity.…”

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

“…21 However, care must be taken to avoid introduction of artifacts during sample processing, 22-24 and, although high-throughput (HTP) approaches have been reported, 24 these methods are not currently routine and require substantial automation, as well as MS instrumentation and expertise. Alternative HTP methods for deamidation detection are typically indirect, for example, reverse-phase HPLC to detect changes in polarity and hydrophobicity, and ion exchange chromatography or electrophoresis based methods to detect changes in charge.…”