Generation of polyethylene glycol (PEG) modified ADA
Calf intestine ADA (Roche Applied Sciences, Indianapolis, IN) was diluted to 500 U/ml in 1 ml of sterile PBS, pH 9.0. The diluted ADA was
dialyzed for 16 h in 2 changes of 500 ml of PBS, pH 9.0. Next, 100 mg/ml
mPEG-SPA, molecular mass 20,000 Da (Nektar Therapeutics, Huntsville,
AL) was added, and the solution was mixed at room temperature for 5 h.
Pegylation efficiency was assessed by zymogram analysis as described (30,
31). Endotoxin was removed using AffinityPak Endotoxin Columns
(Pierce, Rockford, IL). PEG-ADA activity was determined by spectrophotometric ADA enzyme assay (32), and diluted to a final concentration of
0.25 U/l in PBS, pH 7.4.
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