ISOLATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE ELASTASE BY CHROMATOGRAPHY ON IMMOBILIZED BENZAMIDINE

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.