Covalent fixation of soluble derivatized dextrans to model proteins in low-concentration medium: application to factor IX and protein C.

Factor IX and protein C are zymogens implicated in blood clotting, and an increase in their plasmatic residence time would be of interest for the treatment of the disorders caused by their deficiency. In this context, the conjugation of these proteins to polymers such as modified dextrans could be used to approach the problem. Conjugate formation in concentrated medium ([protein] >50 g/L) is well documented, whereas drastic dilution ([protein] <1 g/L) is quite unfavorable. Before studying the binding of factor IX and protein C to polymers, the coupling of model proteins (human hemoglobin, Hb; human serum albumin, HSA) in low-concentration medium to benzenetetracarboxylate dextran (BTC-dextran) and dialdehyde dextran was investigated. To obtain soluble benzenetetracarboxylate dextran-based conjugates, the conditions of coupling were optimized; the use of sulfo-NHS was necessary to form a conjugate with benzenetetracarboxylate dextran. In fact, the O-acylurea intermediate formed between coupling agent [1-ethyl-3(3-dimethylaminopropyl) carbodiimide, EDC] and BTC-dextran must be stabilized. Concerning dialdehyde dextran, a more oxidized polymer and a higher pH of the buffer of coupling than for highly concentrated solution must be used to obtain a conjugate. Whatever polymer is used, HSA appeared clearly less reactive than Hb, which can be attributed to the better reactivity of N-terminal amino groups in this latter protein and to the marked affinity of benzenetetracarboxylate dextran for it. No soluble conjugate was formed between the same dextran derivatives and factor IX or protein C. Moreover, the activity of both coagulation factors was dramatically decreased by contact with EDC and glutaraldehyde, a small molecule. Thus, bad accessibility of protein amino groups is probably responsible for this lack of reactivity. Nevertheless, it could be shown that carboxylate and amino groups were essential to the activity of factor IX and protein C.