Isolation and partial characterization of an elastase-associated acidic endopeptidase and its interaction with elastase at low ionic strength

Abstract

An acidic endopeptidase has been isolated from dried preparations of swine pancrease by column chromatography on DEAE cellulose. The yield of acidic endopeptidase from 100 g of pancreas powder was 210 mg. The product was found to be electrophoretically homogeneous and produced a single peak in the analytical ultracentrifuge. The sedimentation coefficient (s 0 20,w) , diffusion coefficient (D 20,w) , apparent molecular weight, electrophoretic mobility, E 1 cm 1% at 280 mμ and pH optimum were respectively, 3.13, 8.7 · 10 −7 cm 2 · sec −1 , 35 300, −5.9 · 10 −5 cm 2 · V −1 · sec −1 , 14.6 and 10.7. The enzyme was inhibited by diisopropyl phosphorofluoridate, by swine serum and to a lesser extent by human serum. It was not “activated” by 1 · 10 −3 M FeCl 2 , MnCl 2, MgCl 2, CaCl 2 or CoCl 2 or by 1 · 10 −3 M ZnSO 4 ; however, the activity of the enzyme was increased by 1 · 10 −3 M cysteine. The similarity of the acidic endopeptidase to proteolytic enzymes described by other investigators is discussed. Gradient elution ion-exchange chromatography of water insoluble elastase euglobulin produces two major water soluble components, elastase and the acidic endopeptidase. When distilled water solutions of the purified enzymes were mixed complex formation occurred (precipitation) between the basic protein elastase and the acidic endopeptidase. The formation of the euglobulin when acid extracts of swine pancreas are dialyzed against distilled water is thus explained. Complex formation was maximal at low ionic strength when the enzymes were oppositely charged and was minimal below the isoelectric point of the acidic endopeptidase (∼ pH 4.1) or above the isoelectric point of elastase (pH 9.5). The reaction was essentially abolished at I 0.011. The equivalence point for the reaction was 2.8 mg of elastase  1 mg of acidic endopeptidase. One mole of acidic endopeptidase is capable of binding approximately four moles of elastase. Although trypsin, another basic protein, was precipitated by the acidic endopeptidase the data suggest a preferential reaction of the latter enzyme with elastase.