Isolation of human polymorphonuclear leukocyte elastase by chromatography on immobilized benzamidine.
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.
Liau
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DF
Department of Pathology, St. Luke's-Roosevelt Hospital Center, Columbia Univ., College of Physicians and Surgeons, New York, NY 10025.
Yin
N X
NX
Ryan
S F
SF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001550:Benzamidines; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000097:blood* / Q000737:chemistry
D000596:Amino Acids; D001550:Benzamidines; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; C032157:benzamidine