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Several prototypes of aromatic (Ar) and non-aromatic (NoAr) cation-exchange ligands suitable for capture of proteins from high conductivity (ca. 30 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal cation-exchangers were found by screening a diverse library of multi-modal ligands and selecting cation-exchangers resulting in elution of test proteins at high ionic-strength. Candidates were then tested with respect to breakthrough capacity of bovine serum albumin (BSA), human IgG and lysozyme in buffers adjusted to a high conductivity. By applying a salt-step or a pH-step the recoveries were also tested. We have found that aromatic multi-modal cation-exchanger ligands based on carboxylic acids seem to be optimal for the capture of proteins at high-salt conditions. Experimental evidence on the importance of the relative position of the aromatic group in order to improve the breakthrough capacity at high-salt conditions has been found. It was also found that an amide group on the alpha-carbon was essential for capture of proteins at high-salt conditions. Compared to a strong cation-exchanger such as SP Sepharose Fast Flow the best new multi-modal weak cation-exchangers have breakthrough capacities of BSA, human IgG and lysozyme that are 10-30 times higher at high-salt conditions. The new multi-modal cation-exchangers can also be used at normal cation-exchange conditions and with either a salt-step or a pH-step (to pH-values where the proteins are negatively charged) to accomplish elution of proteins. In addition, the functional performance of the new cation-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 10 days. For BSA it was also possible to design cation-exchangers based on non-aromatic carboxyl acid ligands with high capacities at high-salt conditions. A common feature of these ligands is that they contain hydrogen acceptor groups close to the carboxylic group. Furthermore, it was also possible to obtain high breakthrough capacities for lysozyme and BSA of a strong cation-exchanger (SP Sepharose Fast Flow) if phenyl groups were attached to the beads. Varying the ligand ratio (SP/Phenyl) could be used for optimizing the function of mixed-ligand ion-exchange media.

Microsomes of developing soya bean cotyledons transfer oleate from oleoyl-CoA to phosphatidylcholine (PC) by two different mechanisms: one in which oleate transfer is accompanied by the release of free CoA and another which results in the exchange of oleate from oleoyl-CoA for unsaturated 18-carbon fatty acids of PC. The acyl exchange can be demonstrated only when bovine serum albumin is present in the incubation medium. ATP-dependent acyl-CoA synthetase is not involved in the exchange process, which apparently does not require any cofactors. In light of this exchange process, the oleate desaturase system was reinvestigated in order to determine what the actual substrate for this system is. Upon incubation of microsomes with high concentrations of [14C] oleoyl-CoA, bovine serum albumin and NADH, it could be conclusively demonstrated that most oleic acid is desaturated while part of the PC molecule. The amounts of [14C]linoleoyl-CoA formed could be explained entirely by the acyl exchange. The physiological significance of the acyl exchange system is discussed. A new method for separation of acyl-CoA from other lipids and free CoA using reversed phase column chromatography also is described.

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