ASTM_E_1759_-_95_2003.pdf

Designation:E 1759 95(Reapproved 2003)Standard Test Method forIsoaspartic Acid in Proteins:Method for the Determinationof Asparagine Deamidation Products1This standard is issued under the fixed designation E 1759;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.INTRODUCTIONThe storage of proteins in aqueous solutions often results in the formation of isoaspartic acidlinkages within the polypeptide chain as a result of the deamidation of aspargine residues and therearrangement of aspartic acid linkages.This test measures the amount of isoaspartic acid residues ina protein or peptide solution by the use of the enzyme protein isoaspartyl methyl transferase andradioactive S-adenosyl-L-methionine.1.Scope1.1 This test method covers the determination of isoasparticacid residues in a protein or peptide sample.This test methodis applicable for the determination of isoaspartic acid residuesin a sample in the range of 2.550 mol/L.Higher concentra-tions can be determined following dilution.The reported lowerrange is based on single-operator precision.1.2 The values stated in SI units are to be regarded as thestandard.1.3 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 isoaspartic acid residueindicates an aspartic acidresidue in which linkage of the polypeptide chain takes placethrough the gamma carboxyl group of the aspartic acid versusthe alpha carboxyl group that is used in the normal peptidelinkage.3.Summary of Test Method3.1 The basis of the procedure given in this test method isthe production of radioactive methanol equal to the amount ofisoaspartic acid residues present in a protein sample throughthe action of the enzyme protein isoaspartyl methyl transferaseand radiolabelled S-adenosyl-L-methionine,a radiolabelledform of a co-factor that is consumed in the enzymatic reactionof the enzyme.During the test a radiolabelled intermediate isformed through the transfer of the labeled methyl group fromS-adenosyl-L-methionine to the alpha carboxy group of isoas-partic acid.This methylated intermediate is then degraded toliberate the methyl group as methanol.The methanol is thencaptured in a methanol diffusion procedure and counted.3.2 A sample of protein is incubated with the enzymeprotein isoaspartyl methyl transferase and radiolabelledS-Adenosyl Methionine in a buffer that results in the accumu-lation of the methyl esters of isoaspartic acid residues throughthe enzymatic transfer of the methyl group from S-adenosyl-L-methionine to isoaspartic acid sites in the protein.Theprotein solution is then treated with a basic solution containingsodium dodecyl sulfate in order to inactivate the enzyme andconvert the methylated isoaspartic acid residues to a succin-imide and free methanol.The methanol is then separated fromthe protein solution through the diffusion of the methanol to ascintillation fluid solution.The methanol transferred to thescintillation fluid is then determined by counting of theradioactivity in the scintillation fluid.4.Significance and Use4.1 Isoaspartic acid residues are generated during incuba-tion of proteins under a wide variety of conditions in aqueoussolution.Such residues are generated most commonly throughthe deamidation of aspargine residues although some reports ofisoaspartic acid formation through the rearrangement of aspar-tic acid residues have been published.4.2 The presence of such residues can indicate that theprotein containing such residues has suffered damage that mayaffect the biological activity of the protein.The precise1This test method is under the jurisdiction of ASTM Committee E48 onBiotechnology and is the direct responsibility of Subcommittee E48.02 on Charac-terization and Identification of Biological Systems.Current edition approved Oct.10,1995.Published December 1995.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.correlation between the level of isoaspartic acid content and thebiological activity of the protein needs to be determined on acase by case basis.4.3 The test measures the level of isoaspartic acid content ina protein sample.This level will often be correlated with thedegree to which the protein has suffered deamidation atasparagine residues.In addition,isoaspartic acid residues canarise on occasion through the rearrangement of aspartic acidresidues.For these reasons,the level of isoaspartic acidresidues in proteins can be used as a general indication that theprotein sample