PROPERTIES OF THE COMPLEX BETWEEN ou-MACROGLOBULIN AND THE PROTEINASE BRINASE FROM ASPERGILLUS ORYZAE

Brinase, a proteinase from Aspergillus oryzae, has previously been shown to have a significant thrombolytic effect in patients suffering from peripheral arterial disease. Brinase is rapidly bound to α₁-proteinase inhibitor and α₂-macroglobulin (α₂M) in plasma. Since binding of a proteinase to α₂ M only results in ste-rical blockingof the active siteand not complete inactivation of the enzyme, it has been suggested that brinase in complex with α₂M may retain ability to digest fibrin.To test this hypothesis, we have characterized the binding of brinase to α₂M and the properties of the complex formed. Analyses by several techniques showed that one molecule of α₂M can maximally bind twomolecules of brinase. The bait region and the thioester bonds of α₂M were cleaved in this reaction in a similar manner as in the reactionwith trypsin. Moreover, a conformational change highly similar to that-caused by trypsin was induced in oα₂M by brinase, as shown by changes of fluorescence emission, far-u.v. circular dichroism and u.v. absorption difference spectra. Thus, brinase appears to bind to α₂M in the same manner as other proteinases. The activities of free brinase and two forms of brinase-α₂M complex, produced by reaction of thetwo components in an equimolar ratio or by saturation of inhibitor with enzyme, were compared by two different assays with high-molecular-weight substrates, i.e. hide powder azure or fibrin. The complex formed with equimolar amounts of brinase and α₂M showed ∼15% of the activity of free brinase in both assays, whereas the complex formed at saturation of inhibitor with enzyme showed ∼35% of the free brinase activity. Although the brinase-α₂M complex, like other α₂M proteinase complexes, is eliminated rapidly from plasma, the anility of the complex to cleave high-molecular-weight substrates may be an explanation of the thrombolytic effect of brinase seen in patients.