A microassay for proteases using succinylcasein as a substrate

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Proteases are crucial enzymes involved in numerous biological processes.
• Accurate and sensitive assays are essential for studying protease activity.
• Existing methods may lack throughput or sensitivity for certain applications.

Purpose of the Study:

• To develop a novel, sensitive photometric assay for quantifying protease activity.
• To adapt the assay for high-throughput screening using microtiter plate format.
• To demonstrate the assay's applicability to biological samples.

Main Methods:

• A chemically modified casein substrate with succinylated amino groups was synthesized.
• Proteolytic hydrolysis was quantified using trinitrobenzene sulfonate (TNBS) to detect newly formed amino groups.
• Assay performed in microtiter plate wells, with absorbance measured by a microtiter plate reader.

Main Results:

• The assay demonstrated high sensitivity in detecting substrate hydrolysis by various proteases (trypsin, chymotrypsin, thermolysin, subtilisin).
• The microtiter plate format allowed for simultaneous measurement of multiple samples.
• The method was successfully applied to measure proteolytic activity in Tapes philippinarum protein extracts.

Conclusions:

• A robust and sensitive photometric assay for protease activity has been established.
• The microtiter plate-based assay facilitates high-throughput analysis of protease activity.
• This method provides a valuable tool for biochemical and proteomic studies, including environmental sample analysis.