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Solid phase bovine thrombin. Preparation and properties

S S Harwig, J L Lee, L A Sherman

Biochimica Et Biophysica Acta

|June 7, 1976

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A novel solid-phase thrombin was developed, retaining significant esterase activity but reduced proteolytic function. This method offers a simple, rapid approach for studying thrombin interactions and developing diagnostic tools for thrombosis.

Area of Science:

Biochemistry
Enzyme immobilization
Protein chemistry

Background:

Thrombin (EC 3.4.21.5) plays a crucial role in hemostasis.
Developing immobilized enzymes is key for biochemical research and diagnostics.
Previous methods for solid-phase thrombin preparation resulted in significant activity loss.

Purpose of the Study:

To develop a new method for preparing solid-phase thrombin.
To characterize the activity and kinetic properties of the immobilized thrombin.
To evaluate the potential applications of the novel solid-phase thrombin.

Main Methods:

Conjugation of thrombin to a glass support with an active ester of N-hydroxysuccinimide under mild conditions.
Assay of specific esterase and proteolytic activities.

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Determination of apparent Michaelis-Menten constants (Km) for both activities.

Main Results:

Immobilized thrombin retained 50% of its specific esterase activity with an identical Km (5 mM).
Only 6% of specific proteolytic activity was retained, with a higher Km (67 muM) compared to soluble thrombin (12.5 muM).
The preparation method was rapid, simple, and superior to previous diazocoupling techniques.

Conclusions:

The new method yields solid-phase thrombin with preserved esterase activity but reduced proteolytic function.
Potential applications include studying fibrinogen/fibrin interactions and developing radiolabeled fibrin for thrombosis detection.
The observed activity loss is likely due to steric hindrance or charge alterations.