THE INFLUENCE OF L-LYSINE ON FACTOR VIII ELUTED FROM MATRIX BOUND DEXTRAN SULPHATE

Abstract

Factor VIII/vWF displays high affinity for matrix bound sulphate groups. Linear salt and pH gradient elution from Dextran Sulphate Sepharose at 4°C resulted in complete separation of the complex from major protein contaminants with typical yields of 70-90% vWF:Ag and vWF (RCoF). Elution in physiological calcium concentrations gave VIII and VIII:Ag yields of 35% and 65% respectively. Addition of L-Lysine (1.0M) to all buffers inhibited VIII/vWF binding, although lysine gradients (0-1.0M) gave comparable binding and elution profiles as C-1.0M NaCl, but with impaired resolution between protein moieties. However, in the presence of L-Lysine, yields of VIII and VIII:Ag were significantly improved to 80% and 100% respectively when assay standardizations with appropriate lysine concentrations were performed. Furthermore, the binding of fibrinogen could be inhibited by 0.15M L-Lysine, 0.075M NaCl in the equilibrating buffer.The presence of enzymic activity, as assessed by S-2222 and S-2238 in the eluting fractions could be abolished by the application of recryoprecipitated material pre-adsorbed with Al(OK)3 and celite. Incorporation of lysine to the buffers with the associated increase in yields further supports the potential viability of the separation principle for large scale purification of Factor VIII.