A method for the chemical modification of y-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 lyophihzed state at 1 10°C this procedure is carried out in solution at 37°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 y-methyleneglutamic acid (r-MGlu). The extent of modification is controlled by the relative amounts of modification reagents to protein. A loo-fold molar excess of reagents to fragment 1 produced a protein molecule containing two y-MGlu residues, while a modification run at lO,OOO-fold molar excess of reagents to protein yielded fragment I containing eight y-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 I,8 y-MGlu fragment 1, and 2 y-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 alhnity for these antibodies; however, the 2 y-MGlu fragment 1 exhibited a moderate affinity and the 8 y-MGlu fragment 1 did not bind to these antibodies. This is in agreement with the current understanding of vitamin K-dependent coagulation proteins which links calcium-dependent behavior to the presence of Gla residues.