FIG 1.

In vitro stability in serum and elimination of rEnvD from the circulation of BALB/c mice. (A) Stability of rEnvD in BALB/c mouse serum. Enzyme (100 nM) was incubated at 37°C in serum, and activity was determined by FRET assay (12). Enzyme activity in relative fluorescence units was converted to concentration of active enzyme by comparison to a standard curve. Error bars represent the range of three separate determinations; the t_1/2 in serum of rEnvD was 2.95 h (177 min). (B) rEnvD in serum obtained from mice intravenously dosed with 10 mg/kg rEnvD. Serum was obtained by terminal bleed, and enzyme activity was determined by FRET assay. Serum concentration (Cp) of rEnvD was obtained by comparison to a standard curve. Error bars represent the range of three separate determinations performed in duplicate. The half-life, t_1/2, was calculated by determination of the elimination rate constant (K_e) and transformation of data to the natural log (ln) to produce a line of best fit for each phase, with the slope equal to K_e: t_1/2 = ln (2)/K_e. The t_1/2 was 0.22 h (13 min) for the initial alpha phase between 0.5 h and 1 h, and it was 2.71 h (163 min) for the beta phase between 2 h and 24 h.