Isolation and partial characterization of rabbit plasma alpha1-antitrypsin

Area of Science:

• Biochemistry
• Proteomics

Background:

• Alpha1-antitrypsin is a key protease inhibitor found in plasma.
• Understanding its heterogeneity is crucial for comprehending its biological functions.

Purpose of the Study:

• To isolate and characterize alpha1-antitrypsin from rabbit plasma.
• To investigate the physico-chemical properties and heterogeneity of rabbit alpha1-antitrypsin forms.

Main Methods:

• Isolation using salting out, ion-exchange chromatography, and affinity chromatography.
• Characterization via crossed immunoelectrophoresis, polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, and isoelectric focusing.
• Separation of major forms (F and S) by preparative PAGE.

Main Results:

• Purified alpha1-antitrypsin showed homogeneity by immunoelectrophoresis but migrated as two broad bands on alkaline PAGE, with subcomponents observed.
• Two major forms, F and S, were separated and found to have identical elution volumes (MW 58,000) and react with trypsin at a 1:1 molar ratio.
• Isoelectric focusing revealed multiple bands for each form (pH 4.4-4.9), indicating microheterogeneity. Both forms share N-terminal glutamic acid and similar compositions.

Conclusions:

• Rabbit alpha1-antitrypsin exists in at least two major forms (F and S) with distinct electrophoretic and isoelectric focusing patterns.
• Despite microheterogeneity, the core physico-chemical properties, including molecular weight and N-terminal amino acid, are conserved between these forms.