Characterization and inhibition of corneal collagenase.

This investigation shows that corneal collagenase operates optimally at a physiologic pH and not at 4.0 or above 8.4, and that the enzyme cleaves the collagen molecule into % and % length fragments. Studies on the dynamics of collagenase inhibition show that the enzyme is calcium dependent. Many potential inhibitors were tested. The collagenase inhibitors sodium and calcium ethylenediaminetetraacetic acid (EDTA) probably inhibit collagenase by removing the essential calcium. They were found to be completely reversible. The inhibition of corneal collagenase by cysteine was partially reversible by the addition of calcium, indicating that part of its inhibition is due to chelatng. However, more than 50 per cent of cysteine s inhibition is irreversible. Dithiothreitol was also found to inhibit collagenase. Iodoacetate was added to dithiothreitol to alkylate its sulfhydryl groups, and then it did not inhibit collagenase. These results indicate that in addition to inhibiting by chelating calcium, cysteine irreversibly inhibits by reducing a disulfide bond in the enzyme molecule. Finally, guanidinium-extracted corneal proteoglycan was shown to prevent collagenase from breaking down collagen.