A fluorometric assay for HIV-protease activity using high-performance liquid chromatography.

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Quantifying in vitro HIV-protease activity is crucial for drug development and understanding viral replication.
• Existing methods may lack the sensitivity, speed, or reproducibility required for high-throughput screening or detailed kinetic studies.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and reproducible method for quantifying in vitro HIV-protease activity.
• To utilize fluorescence detection coupled with High-Performance Liquid Chromatography (HPLC) for precise enzyme activity measurement.

Main Methods:

• Developed a method based on the endoproteolytic conversion of N-dansyl-SQ-NYPIV (substrate) to N-dansyl-SQNY (product).
• Employed N-dansyl group as a fluorescence label, assessing its minimal impact on kinetic parameters.
• Utilized a single, rapid (3-minute) isocratic reverse-phase HPLC separation with fluorescence detection.

Main Results:

• Achieved sensitive detection of as low as 0.2 pmol of product.
• Demonstrated high reproducibility of the assay.
• Confirmed that the N-dansyl label did not significantly alter peptide-enzyme kinetic parameters.

Conclusions:

• The developed HPLC-based fluorescence assay provides a rapid and sensitive method for quantifying in vitro HIV-protease activity.
• The assay is highly reproducible and suitable for analyzing large sample numbers and conducting rigorous enzymological studies.
• This method offers a valuable tool for HIV research, including drug discovery and mechanistic studies.