Measurement of alligator (Alligator mississippiensis) serum angiotensin converting enzyme (ACE) using a miniaturized colorimetric assay

Oftentimes enzyme assays can be difficult when utilizing tissues from rare, endangered or exotic animals due to limited tissue supplies. We have miniaturized a common assay for angiotensin converting enzyme (ACE) to a microtiter plate format. The assay is based on the reduction of the optical density at 340 nm when the substrate 2-furanacryloyl-l-phenylalanylglycylglycine (FAPGG) is enzymatically-converted to the product 2-furanacryloyl-l-phenylalanine (FAP). The assay was used to measure ACE activity in alligator serum. The optimal pH for this assay was 7, while the ionic strength-dependence was optimal at 25–50 mM NaCl. ACE enzyme activity requires zinc, and thus the assay was sensitive to the presence of EDTA. Inclusion of only 0.2 mM EDTA-inhibited ACE activity by more than 70%. The assay only required the use of only 10 μL of sample for measurable activity, and maximal activity was recorded using 60–65 μL of serum, making it ideal for use with small amounts of blood or tissue extract.