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Tissue dissociation enzyme neutral protease assessment.

A G Breite1, F E Dwulet, R C McCarthy

  • 1VitaCyte LLC, Indianapolis, Indiana 46202, USA. abreite@vitacyte.com

Transplantation Proceedings
|August 10, 2010
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Summary

A new fluorescent assay improves neutral protease measurement for human islet isolation. This method offers greater consistency and accuracy than older assays, aiding in the development of better tissue dissociation enzymes.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Cell Biology

Background:

  • Neutral proteases are crucial for human islet isolation but exhibit variable activity.
  • Existing spectrophotometric assays for neutral proteases lack consistency and accuracy.
  • Variability in protease activity impacts the efficacy of tissue dissociation enzymes.

Purpose of the Study:

  • To develop a more reliable and flexible assay for measuring neutral protease activity.
  • To compare the performance of a new fluorescent assay with a traditional spectrophotometric assay.
  • To characterize the specific activities of different neutral proteases used in cell isolation.

Main Methods:

  • Developed a homogeneous, kinetic fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as substrate.
  • Compared the new fluorescent assay with a spectrophotometric endpoint assay using azocasein substrate.
  • Determined steady-state reaction rates and interassay coefficients of variation for Bacillus polymyxa protease and thermolysin.
  • Incorporated enzyme inhibitors to identify contaminants in purified collagenase preparations.

Main Results:

  • The fluorescent assay demonstrated improved consistency with lower interassay coefficients of variation (<9% for B. polymyxa, <15% for thermolysin) compared to the spectrophotometric assay (54% and 36%, respectively).
  • The new assay allowed for accurate determination of specific activities for various neutral proteases, revealing differences in their enzymatic characteristics.
  • Inhibitor studies using the fluorescent assay identified clostripain as a major contaminant in purified collagenase preparations.

Conclusions:

  • The developed fluorescent microplate assay provides a more sensitive, flexible, and reproducible method for assessing neutral protease activity in tissue dissociation enzymes.
  • This improved assay methodology is critical for optimizing enzyme preparations used in human islet isolation.
  • Understanding the specific activities and contaminants of neutral proteases is essential for advancing cell isolation procedures.