Affinity Chromatography of Serum Albumin with Fatty Acids Immobilized on Agarose

Abstract Fatty acids immobilized on agarose have been employed in the isolation and study of serum albumin by affinity chromatography. Various oleyl- and palmityl-aminoalkyl-amino-agarose preparations bound about 10 mg of albumin per ml of agarose; other proteins were retained in smaller amounts and with lesser affinity. Fatty acid-agarose columns which had been exposed to human serum and then washed yielded essentially pure albumin upon elution with 50% alcohol at pH 3. Lengthening the aminoalkylamino arm from 2 to 10 carbon atoms had little effect on the capacity for albumin, but increased the binding of other serum proteins. That the albumin binding was caused by the immobilized fatty acids was shown by the use of control preparations without fatty acids, by the inverse relation of albumin binding to the fatty acid content of the albumin, and by the ability to elute albumin from the agarose with solutions of sodium oleate. Efforts were made to isolate fatty acid binding regions of the bovine albumin molecule after tryptic digestion of albumin which was bound to fatty acid-agarose. Two peptides with molecular weight of about 10,000 and 23,000 were obtained which were resistant to further digestion; the larger of these was purified and its amino acid composition determined. This tryptic peptide lacked cysteine and tryptophan, and apparently arises from the carboxyl-terminal portion of the albumin molecule. Some implications on the properties of the fatty acid-binding sites on albumin have been offered.