ESR study of free and immobilized elastase

Area of Science:

• Biochemistry
• Enzyme immobilization
• Protein conformational analysis

Background:

• Porcine pancreatic elastase is a key enzyme with applications in various fields.
• Understanding enzyme behavior upon immobilization is crucial for developing efficient biocatalysts.
• Electron spin resonance (ESR) spectroscopy offers a sensitive method for probing enzyme structure and dynamics.

Purpose of the Study:

• To investigate the structural and dynamic changes of porcine pancreatic elastase upon immobilization on polyacrylamide beads.
• To assess the impact of immobilization on the enzyme's catalytic activity and thermal stability.
• To utilize ESR spectroscopy with a spin label probe to analyze the microenvironment of the active site.

Main Methods:

• Enzyme immobilization using glutaraldehyde on polyacrylamide beads.
• Conformational and dynamical analysis using a nitroxide spin label (1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate) targeting the serine-195 residue.
• Electron spin resonance (ESR) spectroscopy for lineshape analysis and copper(II) ion quenching studies.
• Assessment of thermal denaturation and reversibility.

Main Results:

• Immobilization on polyacrylamide beads retained significant catalytic activity.
• ESR studies indicated restricted, pH-dependent mobility of the spin label, largely unaffected by immobilization, suggesting no major conformational changes near the active site.
• Copper(II) ion binding distance to the nitroxide probe remained consistent before and after immobilization.
• Immobilized elastase showed 70% reversibility upon thermal denaturation, unlike the irreversible denaturation of the free enzyme, though the denaturation temperature was unchanged.

Conclusions:

• Immobilization of porcine pancreatic elastase on polyacrylamide beads is an effective strategy that preserves catalytic function and active site integrity.
• The enzyme exhibits enhanced thermal reversibility upon immobilization, a beneficial property for industrial applications.
• ESR spin labeling provides valuable insights into the conformational dynamics of immobilized enzymes.