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Isolation of human polymorphonuclear leukocyte elastase by chromatography on immobilized benzamidine
1Department of Pathology, St. Luke's-Roosevelt Hospital Center, Columbia Univ., College of Physicians and Surgeons, New York, NY 10025.
Abstract:
Recent evidence suggests that polymorphonuclear leukocyte (PMN) elastase causes tissue injury in a variety of diseases. Current methods of purification of elastase involve several steps which result in a low yield. We report a simple purification method. PMN (10(9) in 4 ml of 0.05 M Tris, pH 7.8, containing 0.2% Triton X-100 were disrupted and homogenized by freezing and thawing followed by sonication. After centrifugation at 100,000 g for 20 min, enzyme was extracted from the pellet with 2.5 ml of 0.05 M Tris/1M NaCl (pH 7.8). The centrifugation-extraction cycle was repeated 3 times. Elastase from 10(8) PMN was then purified using a 1 ml Protease Inhibitor Affinity-Filter prepared by binding benzamidine to silica. Enzyme activity was determined by cleavage of the synthetic substrate N-Suc-(Ala)3-pNa. SDS-PAGE demonstrated 2 polypeptides, molecular masses of 29 and 27 kD with amino acid composition and partial N-terminal sequence (Ile-Val-Gly-Gly-Arg-Arg-Ala-Arg-Pro-His-Ala-Trp-Pro-) identical with those previously reported for elastase. We obtained 50 micrograms elastase (34-fold purification) with specific activity of 52 U/mg/min from 10(8) PMN. This represents a much greater recovery (23% yield) than is achieved by other methods. This method is simple, highly reproducible, and can be performed within a 2-day period.
Insights
This study presents a simplified method for purifying polymorphonuclear leukocyte (PMN) elastase, yielding higher recovery and specific activity. This advancement offers a more efficient way to obtain PMN elastase for research and potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Polymorphonuclear leukocyte (PMN) elastase is implicated in tissue injury across various diseases.
- Existing purification methods for PMN elastase are multi-step, leading to low yields.
- A need exists for a more efficient and reproducible purification technique.
Purpose of the Study:
- To develop a simple and efficient method for purifying polymorphonuclear leukocyte (PMN) elastase.
- To achieve higher yields and specific activity compared to current methods.
- To validate the identity and purity of the obtained elastase.
Main Methods:
- PMN were disrupted via freezing/thawing and sonication.
- Repeated centrifugation and extraction cycles were employed to isolate the enzyme.
- Purification was finalized using a benzamidine-silica affinity filter.
- Enzyme activity was measured using N-Suc-(Ala)3-pNa substrate.
Main Results:
- A 34-fold purification of elastase was achieved from 10^8 PMN.
- Specific activity of 52 U/mg/min was obtained.
- SDS-PAGE confirmed the presence of 29 and 27 kD polypeptides, consistent with elastase.
- A yield of 23% was achieved, significantly higher than previous methods.
Conclusions:
- The developed method provides a simple, reproducible, and efficient means of purifying PMN elastase.
- This method offers a substantially improved yield and recovery.
- The purified enzyme exhibits characteristics identical to previously reported elastase, validating the technique.