Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.
Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.
D015055:1-Carboxyglutamic Acid; D002118:Calcium / Q000494:pharmacology; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005453:Fluorescence; D005557:Formaldehyde / Q000494:pharmacology; D005971:Glutamates; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D013329:Structure-Activity Relationship
D005971:Glutamates; D009025:Morpholines; D005557:Formaldehyde; D015055:1-Carboxyglutamic Acid; C037574:morpholine; D005164:Factor IX; D015949:Factor IXa; D002118:Calcium