[35] Assay and purification of phospholipase A2 from human synovial fluid in rheumatoid arthritis

In rheumatoid arthritis, the synovial fluid contains phospholipase A2 (PLA2) in a soluble form and in relatively high amounts. Several procedures have been developed for purifying PLA2 from rheumatoid synovial fluid, but low yields are reported, with typical recoveries accounting for only 4–8% of the total enzyme activity. This chapter describes a purification protocol that exploits the remarkable stability and solubility of this PLA2 at low pH and results in yields of total enzymatic activity of greater than 50%. With this procedure, approximately 10μg of homogeneous PLA2 is routinely recovered from only 50 ml of joint fluid. The most convenient and sensitive assay for the synovial fluid PLA2 uses [3H]oleate-labeled Escherichia coli cells as the substrate. PLA2 assays are carried out in 200-μA reactions containing 10 mM CaCl2 and 1 mg/ml bovine serum albumin. All of the purification steps are carried out at 4° unless specified otherwise. The procedure may be interrupted, with fractions stored at 4° until further processing. In working with samples after the gel-filtration step, dilutions of column fractions should be made into acetate buffer containing 1 mg/ml bovine serum albumin to prevent loss of enzyme because of surface adsorption. The enzyme is purified over 200,000-fold with an overall yield of 58%. The final preparation exhibited a specific activity of 188 units/mg. The apparent activity of synovial fluid PLA2 shows an unusual dependence on ionic strength that complicates quantitation during purification, as evidenced by the fluctuations observed in total activity units.