Table 1.

Purification table for the predominant human mitochondrial phospholipase A₂

Myocardial mitochondria (200 mg) were isolated from human non-failing human heart, resuspended in 20 mm Na₂CO₃ buffer (pH 11.4, containing 10% glycerol, 2 mm EGTA, and 2 mm EDTA), and homogenized by brief sonication. Phospholipase A₂ activity in the supernatant after ultracentrifugation was purified by multiple chromatographic steps, including DEAE, Mono P chromatofocusing, and Mono Q FPLC through following the release of [¹⁴C]AA from [¹⁴C]PAPC substrate. It should be noted that the initial yield of crude homogenates assigned as 100% is certainly underestimated due to substantial isotope dilution of the radioactive substrate by endogenous lipids present in the mitochondrial homogenate. The amount of protein in the active fractions from each chromatographic step was determined by a Bradford protein assay with BSA as standard. The details for each protein purification step are described under “Experimental procedures.”

Mitochondria (200 mg)	Total protein	Total activity	Specific activity	Purification	Yield
	mg	nmol/min	nmol/min·mg	-fold	%
Crude homogenates	200	0.94	0.0047	1	100
pH 11.4 extraction	62	0.53	0.0085	1.8	53
DEAE	6.8	2.9	0.43	91	306
Mono P	0.5	1.3	2.7	510	138
Mono Q	0.018	1.6	87	18,596	170