Purification and partial characterization of human pancreatic elastase

Area of Science:

• Biochemistry
• Enzymology
• Human Physiology

Background:

• Human pancreatic juice contains various enzymes, including elastolytic proteases.
• Understanding the specific properties of human pancreatic elastase is crucial for digestive physiology and disease research.

Purpose of the Study:

• To isolate and characterize the elastolytic enzyme from human pancreatic juice.
• To determine the enzyme's purity, molecular weight, and enzymatic activity.
• To confirm its identity as the primary elastolytic enzyme in human pancreatic secretions.

Main Methods:

• Enzyme isolation using a multi-step purification process involving Sephadex G-25, SP-Sephadex C-50 ion-exchange chromatography, and Trasylol-Sepharose 4-B affinity chromatography.
• Characterization using analytical disc electrophoresis, dodecylsulfate-electrophoresis, and active site titration.
• Immunodiffusion studies with specific antibodies to confirm enzyme identity.

Main Results:

• A homogeneous elastolytic enzyme was isolated with a 50% yield, representing approximately 10% of the total protein in pancreatic juice.
• The purified enzyme has a molecular weight of 26,300 Da, is cationic, lacks carbohydrates, and exhibits specific kinetic properties (Km for Boc-Ala-ONp = 5.13 X 10(-4) M).
• Immunological studies confirmed this purified enzyme as the sole elastolytic protease in activated human pancreatic and duodenal juice.

Conclusions:

• The study successfully isolated and characterized a key human pancreatic elastase.
• The purified enzyme exhibits distinct biochemical and functional properties compared to porcine elastase, particularly in elastin degradation.
• The findings confirm the enzyme's role as the primary elastolytic protease in human pancreatic secretions.