Purification of urokinase by affinity chromatography

Area of Science:

• Biochemistry
• Protein purification
• Enzymology

Background:

• Commercially available urokinase (EC 3.4.99.26) preparations contain impurities, including unrelated proteins and urokinase degradation fragments.
• These contaminants can affect the enzyme's activity and experimental results.

Purpose of the Study:

• To describe a method for further purifying urokinase.
• To obtain a highly pure urokinase preparation suitable for research and potential therapeutic applications.

Main Methods:

• Chromatography on benzamidine-Sepharose was employed for the purification of urokinase.
• Molecular weight determination of the purified urokinase forms.

Main Results:

• The purification process successfully yielded two distinct molecular forms of urokinase.
• The final preparation contained urokinase with molecular weights of 31,000 and 54,000 daltons.
• The 54,000-dalton form appears to be a dimer, with the 31,000-dalton form representing one of its subunits.

Conclusions:

• Benzamidine-Sepharose chromatography is an effective method for purifying urokinase.
• The purified preparation consists of two active molecular forms, suggesting different functional states or subunits.
• A monospecific antiserum against urokinase was successfully raised, useful for further characterization.