A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Angiotensin I-converting enzyme (ACE) activity is crucial in biological and clinical contexts.
• Existing ACE assays can be time-consuming or reagent-intensive.

Purpose of the Study:

• To develop a sensitive, rapid, and cost-effective fluorimetric assay for ACE activity.
• To adapt the assay for a 96-well plate format for increased efficiency.

Main Methods:

• Utilized hippuryl-L-histidyl-L-leucine as a substrate.
• Quantified the fluorescent adduct of L-histidyl-L-leucine via fluorimetry.
• Adapted the protocol for a 96-well plate format.

Main Results:

• The fluorimetric assay demonstrated sensitivity, speed, and cost-effectiveness.
• The 96-well plate format yielded comparable data to existing assays.
• Apparent Km and kcat values for purified testis ACE were determined as 3.0 mM and 195.7 s(-1).

Conclusions:

• The developed fluorimetric assay is suitable for routine ACE activity measurements.
• The 96-well plate adaptation enhances efficiency for both routine and kinetic analyses.
• The protocol can be completed within 4 hours.