Novel photo affinity cross-linking resin for the isolation of heparin binding proteins

A new photo cross-linking reagent, 2-(4-azidophenylamino)-4-(1-ammonio-4-az abicyclo[2,2,2]oct-1-yl)-6-morpho-lino-1,3,5-triazine chloride (named AA-D) was developed, which was used for the identification of several heparin-bi nding proteins on the surface of intact platelets. Also a functional resin for the isolation of heparin-binding proteins from a protein mixture was pr epared. Heparin was first immobilized onto a polystyrene Merrifield resin or Argo-g el(TM) using a reductive amination reaction. Then the immobilized heparin w as coupled with the AA-D reagent to give a functional resin. The resin was incubated with an individual protein, such as ovalbumin, bovine serum album in (BSA), antithrombin III (ATIII) or a synthetic peptide corresponding to the heparin-binding domain of ATIII, or with a mixture of the above protein s. This was then photo-irradiated to induce the cross-linking between the h eparin and the protein bound to it. Ovalbumin, a non-heparin-binding protei n, was recovered from the supernatant of the irradiated incubation mixture. BSA, known to bind to heparin nonspecifically, was found in the washing of a high ionic strength solution. In contrast, native ATIII and its heparin- binding domain peptide were covalently bound to the heparin on the resin an d liberated from the resin only after degradation of the resin-bound hepari n with nitrous acid or heparinase I treatment. The experiments demonstrated that the resin modified with heparin and the AA-D have a definite practica l value in the selective isolation of heparin-binding proteins.