A simple and sensitive proteinase assay using Sepharose 4B-coupled fluorescamine-labeled casein as a substrate

Area of Science:

• Biochemistry
• Enzymology

Background:

• Proteinase assays are crucial for various biological and diagnostic applications.
• Existing methods may lack sensitivity or require specialized reagents.

Purpose of the Study:

• To develop a simple, sensitive, and accurate method for proteinase assay.
• To utilize fluorescamine-labeled casein-Sepharose 4B as a substrate for enzyme activity determination.

Main Methods:

• Casein was coupled to cyanogen bromide-activated Sepharose 4B at pH 10.0.
• The resulting casein-Sepharose 4B was labeled with fluorescamine at pH 8.0.
• Enzyme activity was measured by quantifying fluorescence (excitation 390 nm, emission 475 nm) in the filtrate after substrate incubation.

Main Results:

• The method demonstrated high sensitivity, detecting as little as 3 ng of trypsin or 10 ng of alpha-chymotrypsin.
• The assay is suitable for proteinases active at neutral to slightly alkaline pH.
• The sensitivity approaches that of radioisotope-labeled protein assays.

Conclusions:

• A simple and sensitive proteinase assay using fluorescamine-labeled casein-Sepharose 4B was successfully developed.
• This method offers a cost-effective and accessible alternative for quantifying proteinase activity.
• The assay's sensitivity and ease of use make it valuable for biochemical research.