FIGURE 5.

Relative fraction of wild-type carboxylase bound to KH₂ at varying KH₂ concentrations. 20 nm fluorescein-labeled human factor IX propeptide in 50 mm MOPS, pH 7.5, 500 mm NaCl, 5% glycerol, 6.3 mm DTT, 66 μm EDTA, 0.16% PC, 0.16% CHAPS, and 0.2% BSA was incubated with 100 nm wild-type carboxylase and varying concentrations of KH₂ at 4 °C and in a final volume of 329 μl, until binding equilibrium was reached. The off rate of the labeled propeptide from carboxylase at each KH₂ concentration was determined by adding unlabeled factor IX propeptide to 3.5 μm and measuring the time-dependent decrease in fluorescence anisotropy. Alkamuls EL-620 was included in the blank with no KH₂.