Phospholipases A₂ (EC 3.1.1.4; PLA₂) hydrolyse the sn-2 ester bonds of sn-3 glycerophospholipids. Expression of the group IIA human secreted phospholipase A₂ (hsPLA₂ gIIA) is induced by bacterial endotoxins and cytokines, and is considered to be a protein of the acute phase immune response showing bactericidal effects against Gram-positive bacteria. However, over-expression of the protein may lead to pathological states, and can contribute to the symptoms observed in autoimmune diseases. We have studied the interaction of suramin with the hsPLA₂ gIIA as a model to study a new type of inhibitor of secreted PLA₂s. Thirteen site-directed charge elimination mutants of the hsPLA₂ gIIA were prepared, expressed in E. coli BL21(DE3){pLysS}, refolded and purified by cationic change chromatography. On binding to the hsPLA₂ gIIA, the suramin intrinsic fluorescence emission at 440nm increases in a sigmoid manner, suggesting a single suramin-binding site on the protein. Binding site mapping as evaluated by the site-directed mutants of hsPLA₂ gIIA revealed that whereas active–site mutants had only marginal effects on suramin binding, the K15A mutant significantly altered this effect suggesting that the suramin binding site is separated from the active–site region.Supported by FAPESP and PRP-USP.