Df. Liau et al., ISOLATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE ELASTASE BY CHROMATOGRAPHY ON IMMOBILIZED BENZAMIDINE, Preparative biochemistry, 23(4), 1993, pp. 439-447
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.