Factors Modulating the Matrix Metalloprotease (MMP) Activity of Dispase

Abstract

Purpose:: The intravitreal injection of Dispase has been shown to induce posterior vitreous detachment in enucleated human eye bank eyes and porcine eyes in vivo due to its function as a specific MMP targeting fibronectin and collagen IV. Herein we explored the factors that influence the MMP activity of Dispase in vitro.

Methods:: Dispase from two commercial sources (GODO Inc and GIBCO Inc) was subjected to electrophoresis in a 4-16% Zymogram (blue casein) gel (Invitrogen) and the result was analyzed by the "Volume Tools" function of Quantity One software (BioRad, 4.0). The activity of dispase was measured by EnzChek protease assay kit E6638 using an ISS Steady-State Fluorometer (Champaign, IL). The AAnalyst 100 atomic absorbance spectrometer (Perkin Elmer) was used to measure the total calcium contained in Dispase. Fura-2, pentapotassium salt, cell impermeant and the calcium calibration buffer kit (Molecular Probes, Inc.) were used to measure free-calcium in the Dispase buffer solution.

Results:: The Dispase from GODO revealed 4.2 fold higher activity than commercially available Gibco dispase on a weight basis. Chelating calcium with EDTA significantly reduced the activity of Dispase. Both GODO (8.5% w/w) and GIBCO (2.5% w/w) Dispase contain bound calcium, and calcium chelation with EDTA significantly reduced the activity of Dispase once the EDTA concentration exceeded 60 µM. The optimum MMP activity was achieved at physiological calcium concentrations of the vitreous (8-10 mM). Paradoxically, calcium concentrations higher than 20 mM also inhibited Dispase activity. The enzyme kinetics on casein showed faster kinetic rate with GODO Dispase (K_d1=0.0043, K_d2=2326.054) compared with GIBCO Dispase (K_d1=0.000, K_d2=615.959). Protein concentration assays revealed that only 14% of the GODO Dispase and 3.2 % of the GIBCO product by weight were proteins. Two other bands at 55.9 and 25 kD revealed MMP activity on zymogram gels in addition to the previously identified MMP at 35.9 Kd. The latter band was responsible for 50.7 ± 3.2% of the MMP activity of the GODO Dispase compared to 30.0 ± 5.2% in the GIBCO product.

Conclusions:: The activity of Dispase can be modulated by environmental calcium concentration. The dispase from GODO is purer and exhibits a higher MMP activity. Removal of impurities within Dispase either by purification or via recombinant production should increase its efficacy.CR: None

Keywords: vitreous • enzymes/enzyme inhibitors • injection