ISOLATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE ELASTASE BY CHROMATOGRAPHY ON IMMOBILIZED BENZAMIDINE

Citation
Df. Liau et al., ISOLATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE ELASTASE BY CHROMATOGRAPHY ON IMMOBILIZED BENZAMIDINE, Preparative biochemistry, 23(4), 1993, pp. 439-447
Citations number
16
Categorie Soggetti
Biology
Journal title
ISSN journal
00327484
Volume
23
Issue
4
Year of publication
1993
Pages
439 - 447
Database
ISI
SICI code
0032-7484(1993)23:4<439:IOHPLE>2.0.ZU;2-A
Abstract
Recent evidence suggests that polymorphonuclear leukocyte (PMN) elasta se causes tissue injury in a variety of diseases. Current methods of p urification of elastase involve several steps which result in a low yi eld. 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 hom ogenized by freezing and thawing followed by sonication. After centrif ugation at 100,000g for 20 min, enzyme was extracted from the pellet w ith 2.5 ml of 0.05 M Tris/1M NaCl (pH 7.8). The centrifugation-extract ion cycle was repeated 3 times. Elastase from 10(8) PMN was then purif ied using a 1 ml Protease Inhibitor Affiny-Filter prepared by binding benzamidine to silica. Enzyme activity was determined by cleavage of t he synthetic substrate N-Suc-(Ala)3-pNa. SDS-PAGE demonstrated 2 polyp eptides, molecular masses of 29 and 27 kD with amino acid composition and partial N-terminal sequence -Val-Gly-Gly-Arg-Arg-Ala-Arg-Pro-His-A la-Trp-Pro-) identical with those previously reported for elastase. We obtained 50 mug elastase (34-fold purification) with specific activit y of 52 U/mg/min from 10(8) PMN. This represents a much greater recove ry (23% yield) than is achieved by other methods. This method is simpl e, highly reproducible, and can be performed within a 2-day period.