A sensitive colorimetric assay for various proteases using naphthyl ester derivatives as substrates.

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Proteases play crucial roles in biological processes.
• Sensitive and convenient assays are needed for protease detection.
• Existing ester derivatives have limitations in sensitivity.

Purpose of the Study:

• To develop a highly sensitive colorimetric assay for various proteases.
• To evaluate alpha-naphthyl ester derivatives as substrates for protease detection.
• To compare the sensitivity and kinetic properties of new substrates with existing ones.

Main Methods:

• Utilized alpha-naphthyl ester derivatives of N alpha-tosyl-L-lysine and N alpha-acetylglycyl-L-lysine.
• Measured the release of alpha-naphthol upon enzymatic hydrolysis.
• Determined minimum detectable concentrations and kinetic parameters (Km) for key proteases.
• Prepared zymograms using the developed substrates.

Main Results:

• The assay demonstrated high sensitivity for trypsin, chymotrypsin, plasmin, thrombin, and urokinase.
• Alpha-naphthyl ester derivatives were more sensitive than methyl or ethyl ester derivatives.
• Lower Km values were observed for alpha-naphthyl esters, indicating higher substrate affinity.
• Zymograms were readily prepared for trypsin and chymotrypsin.

Conclusions:

• The developed colorimetric assay is convenient and highly sensitive for a range of proteases.
• Alpha-naphthyl ester derivatives represent superior substrates for protease assays and zymography.
• This method offers an improved tool for biochemical and diagnostic applications involving protease activity.