Natural protease inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis.

This research discusses an electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors. Using agarose with denatured fibrinogen and a process involving electrophoretic separation and soaking in a protease solution, this method reliably determines inhibitor activity and enzyme specificity.

Methodology of the Electrophoretic Procedure

• The study describes a unique method to assay protein protease inhibitors, especially beneficial when investigating crude biological materials without specific antisera. The assay uses an electrophoretic procedure.
• The supporting medium used in the procedure is agarose, into which denatured fibrinogen is incorporated as a substrate for proteases.
• This electrophoretic procedure is divided into two steps. The first is the electrophoretic resolution of the inhibitor-containing material, and the second is the detection of the inhibitor bands through their protease inhibiting activity.

Detection and Visualization of Inhibitor Bands

• The positioning of the inhibitor can be made visible as a colored band of denatured fibrinogen, which has avoided digestion by the protease.
• By electrophoretically separating multiple copies of a sample of biological fluid, and then immersing each of them in the solution of a distinct protease, the enzyme specificity of a particular inhibitor band can be accurately identified.

Quantification and Calibration of Protease Inhibitors

• Once the inhibitor bands have been made visible, they can then be quantitated using densitometry in selected cases. The inhibitor activity can be accurately calculated through this process.
• A reference serum calibrated with Trasylol serves as a standard for determining the inhibitor activity. An application of this technique is demonstrated in the paper by reporting the activity of the alpha-1-protease inhibitor in healthy horses.