A method for specific chemical modification of gamma-carboxyglutamic acid residues in proteins.
A method for the chemical modification of gamma-carboxyglutamic acid (Gla) residues in proteins is introduced that has the combined advantages of mildness, a high degree of specificity, and the ability to introduce a radiolabel at modification sites for ease in quantitation. Unlike other Gla modification procedures which are performed in the lyophilized state at 110 degrees C, this procedure is carried out in solution at 37 degrees C. The addition of morpholine and formaldehyde to a slightly acidic solution of bovine prothrombin fragment 1 (residues 1-156) results in the conversion of Gla residues to gamma- methyleneglutamic acid (gamma- MGlu ). The extent of modification is controlled by the relative amounts of modification reagents to protein. A 100-fold molar excess of reagents to fragment 1 produced a protein molecule containing two gamma- MGlu residues, while a modification run at 10,000-fold molar excess of reagents to protein yielded fragment 1 containing eight gamma- MGlu residues per molecule. The specificity of this modification is illustrated by the interaction of native and modified protein with antibody populations directed against fragment 1. Native fragment 1, 8 gamma- MGlu fragment 1, and 2 gamma- MGlu fragment 1 show fairly similar behavior toward whole anti-fragment 1 serum. Differential behavior was exhibited by the native and modified proteins toward a subpopulation of antibodies specific to the calcium ion conformation of fragment 1. Unmodified fragment 1 displayed a strong affinity for these antibodies; however, the 2 gamma- MGlu fragment 1 exhibited a moderate affinity and the 8 gamma- MGlu fragment 1 did not bind to these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis; D000917:Antibody Formation; D000936:Antigen-Antibody Complex; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003653:Decarboxylation; D005971:Glutamates / Q000032:analysis*; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D011498:Protein Precursors; D011506:Proteins; D011516:Prothrombin / Q000276:immunology; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D005971:Glutamates; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; D015055:1-Carboxyglutamic Acid; C030977:4-methyleneglutamic acid; C029733:prothrombin fragment 1; D011516:Prothrombin