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CAROL WELLER HOOK, F. GEOFFREY BULL, VICTORIA IWANIJ, STUART I. BROWN; Purification of Corneal Collagenases. Invest. Ophthalmol. Vis. Sci. 1972;11(9):728-734.
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Collagenases from alkali-burned and inflamed corneas were purified by (NH4)2SO4 gel filtration and ion exchange chromatography. After purification, the collagenase of the inflamed cornea appeared as one homogenous band in polyacrylamide gel disc electrophoresis. This collagenase had a molecular weight of 65,000, an isoelectric point of 7.0, and was inhibited by cysteine, dithiothreitol (DTT), Na2EDTA, Ca Na2EDTA and freshly prepared rabbit serum. When n-ethylmaleimide was added to collagenase that had been inhibited by DTT, the inhibition could not be reversed by oxidation indicating that a disulfide linkage was essential to enzyme activity. Purification of the collagenase from the alkali-burned cornea on G-200 Sephadex showed two active enzyme peaks. The smaller and less active collagenase had a molecular weight of 65,000 and the larger collagenase had a molecular weight greater than 158,000. It was theorized that the larger collagenase may be either an aggregate of the smaller collagenase or may represent a distinct collagenase. Both enzymes were inhibited by Na2EDTA and cysteine. The purified collagenases cleaved collagen into breakdoion products that are characteristic of most animal collagenases.
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