The binding of interleukin 2 to heparin revealed by a novel ELISA method.

An increasing number of polypeptide growth factors and cytokines are now known to bind to heparin and heparan sulphate glycosaminoglycans. Well studied examples are members of the chemokine and fibroblast growth factor families. However other growth factors including proinflammatory and haematopoietic cytokmes also bind to heparin [ 1-41, Such interaction with glycosaminoglycans of the extracellular matrix and cell surface may be important in protecting cytokines against degradation, and in localising them close to their sites of secretion. We have developed a novel ELISA approach for the investigation of heparin-cytokine interactions. For this heparin is covalently bound via its reducing terminus to a protein carrier, bovine serum albumin (BSA), using sodium cyanoborohydnde. This coupling method is intended to ensure that the heparin chains remain accessible, for subsequent engagement in protein-binding interactions. Antithrombin 111 (AT 111) and fibroblast growth factor-2 (basic-FGF), both proteins with well characterised heparinbindmg properties, bind to the heparin-BSA complex in our ELISA. In both cases, bindmg is a dose dependent and saturable over the 0-3Ong range. Free heparin competes with binding of both proteins to the complex, with an EC,, value of around 50Ong/ml. With AT III, we have found that an AT 111-binding heparin fiaction separated by aflimty chromatography on immobilised AT 111, is a strong competitor in our ELISA. Conversely the low affinity heparin fraction is a poor competitor. Thus our ELISA shows the anticipated specificity in heparin-protein interactions. We have now shown that recombinant human i n t e r l e h 2, rIL-2, binds to the heparin complex (see Fig 1). The binding is dose dependent, but over a range of 0-100ng does not reach saturation. As with AT 111 and FGF-2, free heparin displaces rIL-2 fiom the complex, but with rIL-2 the EC,, for mhibition of binding is an order of magnitude higher, at around 7pg/ml. Thus it would appear that the afhuty of IL-2 for heparin is lower than that of AT 111 and