Conversion of exposed aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues

Abstract

1. 1. The feasibility of converting exposed carboxyl groups in proteins to carboxamido groups using 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) as a condensing agent was demonstrated. 2. 2. The effectiveness of ammonia as a nucleophile in the EDC-mediated condensation with carboxyl groups in proteins was determined. In 5.5 M NH 4Cl at pH 4.75, 11.5, 14.7, 9.0, 5.0 and 5.1 carboxyl groups are converted to carboxamido groups in chymotrypsinogen A, α-chymotrypsin (EC 3.4.4.5), lysozyme (EC 3.2.1.17), ribonuclease (EC 2.7.7.16) and trypsin (EC 3.4.4.4), respectively. 3. 3. The number of carboxamido groups found was determined from the amount of extra ammonia appearing in acid-hydrolyzed protein which was reacted with EDC and NH 4Cl. In one case, the number of β- and γ-carboxamido groups formed was also determined enzymically by digesting treated protein with proleolytic enzymes and then enzymically assaying the digest for free asparagine and glutamine. Both acid hydrolysis and enzymatic assays gave essentially the same value for the yield of carboxamido groups. 4. 4. 5.5 M NH 4Cl, 1 M glycine methylester, and 1 M glycinamide were all equally effective in modifying carboxyl groups. Since previous studies showed that the glycine derivatives quantitatively modify exposed carboxyl groups in proteins, it was concluded that NH 4Cl may be used to quantitatively convert exposed carboxyl groups in proteins to carboxamido groups. Now that it is possible to convert aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues, it should be possible to establish with greater certainty the role of carboxyl groups in the function and structure of proteins.