A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoproteina] (Lpa]) proceeds in two steps. In the first step, non-covalent interactions between apolipoproteina] (apoa]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apoa]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lpa] particle. Several methods are currently used to study the assembly of Lpa], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lpa]/apoa] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lpa] and apoa] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apoa] were capable of competing with LDL binding. The binding of LDL to Lpa]/apoa] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apoa] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apoa] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lpa] formation.