(416f) Manipulation of the Adsorption of Factor II, a Protein C Homologue, Using Imidazole during Immobilized Metal Affinity Chromatography Purification of Protein C from Cohn Fraction IV-1

Lee, J. J. - Presenter, University of Louisville
Bruley, D. F. - Presenter, University of Maryland Baltimore County
Kang, K. A. - Presenter, University of Louisville

Protein C (PC) has the function of an anticoagulant, antithrombotic, anti-inflammatory, and antiapoptotic in blood plasma. The potential for PC as a therapeutic includes uses for PC deficiency, various thrombo-embolic complications resulting from major surgery or trauma, advanced sepsis, or stroke. PC does not have any known clinical side effects, such as bleeding and skin necrosis that can occur with patients using anticoagulant treatment such as coumadin and heparin. Cohn Fraction IV-1 (CFIV-1) paste is selected as a PC source for this purification study. CFIV-1 is a plasma derivative by-product from a fractionation process for human serum albumin that use to be discarded and has about 100?nmicro-grams of PC/gram-paste. Thus, CFIV-1 is an ideal PC source, but it has a large amount of coagulant proteins that are homologous to PC. Among these coagulants in CFIV-1, Factor II (FII) is the most problematic because of its long half-life and quantity to that of PC. The separation of PC from FII is very critical to make available PC as a therapeutic. PC purification from CFIV-1 is difficult because is requires high specificity to separate PC from the PC homologues. Therefore, immunoaffinity chromatography (IAC) is the current method used to purify PC with monoclonal antibodies, but it is expensive. However, immobilized metal affinity chromatography (IMAC) utilizes metal ions to adsorb proteins with surface histidine residues. In our previous study, the qualitative results by Wu, et al. demonstrated that IMAC had the specificity to separate PC from FII in a mixture. In IMAC, imidazole is an analog to histidine that is frequently used in elution as a competitively adsorbing molecule against histidine. However, it can also be used during adsorption to manipulate protein adsorption by diminishing available metal ions. The adsorption of PC is slightly different than that of FII because PC has fifteen surface histidines and FII only has five surface histidines. Therefore, it is possible to decrease FII adsorption while upholding PC adsorption by using specific amount of imidazole during the adsorption step. Using imidazole during adsorption may also decrease the affinity of the adsorbed FII and increase the efficiency washing FII out of the IMAC column. The separation of PC from FII is examined in this study by controlling the amount of imidazole utilized during adsorption. In batch experiments, of 20 micro-grams of PC were reacted with 100 micro-liters of Cu (II) chelated IMAC matrix at various imidazole. This was also repeated for FII. The amounts of adsorbed PC and FII were quantified, by measuring the amount of free PC and FII in the liquid phase by ELISA. These results demonstrate the effect of how imidazole can decrease the adsorption of FII and provide partial separation of PC from FII during the adsorption stage and improve the purification of PC by IMAC.