FACILE PURIFICATION OF FIBRINOGEN FRAGMENTS USING A COMPUTER-BASED MODEL WITH GENERAL APPLICABILITY TO THE GENERATION OF SALT GRADIENTS

We and others have recently shown that specific fragments of cross-lin ked fibrin affect cell behavior. In order to develop a facile method f or the preparative scale purification of fibrin fragment D dimer, a si mple gradient generating system for conventional chromatography was de veloped and validated, and methods of fibrin fragment D dimer purifica tion were compared. The experimentally measured salt concentration/tim e relationship fell directly on the model-predicted line. Model-predic ted changes in the reservoir volume and/or salt concentration in the l imit buffer affected both the initial slope and the shape of the conce ntration/time relationship. This gradient generation method was used t o separate the D domains of fibrin(ogen) from the amino terminal regio n E domain using anion-exchange chromatography. While the predicted sa lt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by C oomassiestained SDS-PAGE and by immunoblotting. In conclusion, a facil e, user-friendly, computer-based method to predict and generate salt g radients was written and validated by direct experimentation. While fi brinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pi-I-based separation technique, (C) 1998 Academic Press.