[3H]Benzoyl-Phe-Ala-Pro ([3H]BPAP) is frequently used as a substrate for lung or heart microvascular ACE activity, in vivo. It has been reported that binding of [3H]BPAP by Conn fraction V bovine serum albumin (BSA) artifactually limits BPAP hydrolysis by isolated rabbit lung. Since BSA binding is not necessarily predictive of binding of BPAP to native serum proteins, we examined for binding of BPAP in sera from seven New Zealand white rabbits and four beagle dogs. Serum samples (200 μl) were incubated with 202 μl of HEPES-buffered (pH=7.4) BPAP/[3H]BPAP solutions (0.162.5 μM). The bound (B) and free (F) fractions were separated using Millipore 10,000 NMWL ultrafilter units (centrifuged at 2000g for 60 min) A 25 μl aliquot from the original mixture and a 25μl aliquot from the ultrafiltrate were assayed for 3H content. The ratios of B/F and B to total (BAT) fractions were calculated as 0.086 ± 0.077 and 0.075 ± 0.063 for rabbit sera and 0.138 ± 0.073 and 0.118 ± 0.058 for dog sera, respectively, for the entire range of BPAP concentrations used. At trace BPAP concentration (0.1 μM) average B/F and B/T values were 0.052 ± 0.086 and 0.044 ± 0.071 for rabbit sera and 0.089 ± 0.068 and 0.078 ± 0.057 for dog sera, respectively These values were not statistically different from zero. These results indicate that the serum protein binding of [3H]BPAP is low and is unlikely to be a limiting factor in the hydrolysis of [3H]BPAP in vivo.
|Publication status||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology