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H.J. Kaplan, T.H. Tezel, L.V. Del Priore; Purification of the Active Constitutent of Dispase for Vitreopharmacolysis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5454.
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Purpose: To identify and purify the active constituent(s) of Dispase responsible for enzymatic posterior vitreous detachment. Methods: The purity of Dispase I and II were analyzed by 2–D gel electrophoresis. Endotoxin contamination of both preparations was determined using the limulus lysate assay. Enzymatic autodegradation and activity of the constituents was tested with a colorimetric gelatinase assay. An active band was purified using a diffusion column. The ability of the purified protein to induce a posterior vitreous detachment was investigated in vitro using freshly enucleated porcine eyes. Results: Both Dispase I and II contains multiple impurities and are contaminated with endotoxin. The gelatinase activity of Dispase II was significantly lower than Dispase I because of higher amounts of non–protein impurities. The enzymatic activity of both preparations was mainly generated by a 38 kDa matrix metalloprotease that was purified in a gel column. The purified enzyme retained its activity on the gelatinase assay. Using freshly enucleated porcine eyes (n=10), the purified enzyme induced complete (8/10) and partial (2/10) PVD within 15 minutes; whereas 1/10 control eyes that received intravitreal BSS only revealed complete, 2/7 a partial PVD and 7/10 no PVD. The enzymatic activity of the purified protein was greater than crude Dispase I which induced fewer complete (3/10) and partial (6/10) PVDs. Conclusions: Crude Dispase contains endotoxin, which is known to cause intraocular inflammation within 24–48 hours of intravitreal injection in vivo. The active enzyme can be purified and has a higher enzymatic activity that crude Dispase I and II. Advances in the production of the purified protein have the potential to induce a safe enzymatic PVD in vivo.
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