Partial purification of collagenase and gelatinase from human polymorpho- nuclear leucocytes

The separation and further purification of human polymorphophonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem. J. 183, 647-6561, are described. The final preparations yielded collagenase of specific activity 260units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 1251-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors a2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 250C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two to three times more effective in the degradation of these insoluble collagens.