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 lime would be of interest fo r the treatment of the disorders caused by their deficiency. In this c ontext, the conjugation of these proteins to polymers such as modified dextrans could be used to approach the problem. Conjugate formation i n concentrated medium ([protein] >50 g/L) is well documented, whereas drastic dilution ([protein] <1 g/L) is quite unfavorable. Before study ing the binding of factor IX and protein C to polymers, the coupling o f model proteins (human hemoglobin, Hb; human serum albumin, HSA) in l ow-concentration medium to benzenetetracarboxylate dextran (BTC-dextra n) and dialdehyde dextran was investigated. To obtain soluble benzenet etracarboxylate dextran-based conjugates, the conditions of coupling w ere optimized; the use of sulfo-NHS was necessary to form a conjugate with benzenetetracarboxylate dextran. In fact, the O-acylurea intermed iate formed between coupling agent [1-ethyl-3(3-dimethylaminopropyl) c arbodiimide, EDC] and BTC-dextran must be stabilized. Concerning diald ehyde dextran, a more oxidized polymer and a higher pH of the buffer o f coupling than for highly concentrated solution must be used to obtai n a conjugate. Whatever polymer is used, HSA appeared clearly less rea ctive than Hb, which can be attributed to the better reactivity of N-t erminal amino groups in this latter protein and to the marked affinity of benzenetetracarboxylate dextran for it. No soluble conjugate was f ormed between the same dextran derivatives and factor IX or protein C. Moreover, the activity of both coagulation factors was dramatically d ecreased by contact with EDC and glutaraldehyde, a small molecule. Thu s, bad accessibility of protein amino groups is probably responsible f or this lack of reactivity. Nevertheless, it could be shown that carbo xylate and amino groups were essential to the activity of factor IX an d protein C.