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Determination of proteolytic enzymes by flow-injection analysis
P Nicholas1, A Lamy, S Reymond
1Nestlé Research Centre, Nestec Ltd, Lausanne, Switzerland.
Analytical Biochemistry
|January 1, 1991
Summary
This study presents a rapid flow-injection analysis method for quantifying proteolytic enzymes like subtilisin and chymotrypsin. The developed assay offers quick, reproducible results with minimal sample and substrate usage.
Area of Science:
- Biochemistry
- Analytical Chemistry
Background:
- Proteolytic enzymes play crucial roles in biological processes.
- Accurate and efficient quantification methods are essential for enzyme research and diagnostics.
Purpose of the Study:
- To adapt and validate a flow-injection analysis (FIA) method for quantifying proteolytic enzymes.
- To optimize the assay for speed, reproducibility, and minimal reagent consumption.
Main Methods:
- Utilized N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a chromogenic substrate.
- Employed the merging zones technique combined with a washing step in FIA.
- Investigated the influence of substrate concentration on spectrophotometric response for subtilisin and chymotrypsin.
Main Results:
- Achieved results in under 15 seconds per sample.
- Demonstrated high sample throughput (90 samples/hour) with good reproducibility.
- Established linear relationships between peak height and enzyme concentration for both subtilisin and chymotrypsin within specific ranges.
Conclusions:
- The developed FIA method provides a fast, sensitive, and reproducible means for quantifying proteolytic enzymes.
- The assay is suitable for small sample volumes and minimizes chromogenic substrate consumption.
- This technique offers a valuable tool for enzyme analysis in various research and diagnostic applications.