Figure 2.

Gla analysis of in vitro carboxylated carboxylase. The propeptide eluant isolated from r-fIX, r-carboxylase BHK cells (100 pmol, Fig. 1) was chromatographed on Q-Sepharose, in vitro carboxylated, and then TCA precipitated and base hydrolyzed. Samples with or without vitamin K in the in vitro protein carboxylation reaction were processed in parallel. After the amino acid hydrolysate was resuspended in water (25 μl), duplicate aliquots (2 μl) were quantitated for radioactivity and duplicate samples (10 μl) were analyzed by HPLC. Peaks corresponding to gla (0.8 min), asp (1.6 min), and glu (3.5 min) were collected and counted, and the radioactivity (in cpm) is indicated at the bottom of the chromatogram.