Hydrophobic affinity chromatography of proteins

Abstract

1. 1. Chromatographic studies have been made of the affinities of a number of purified proteins for agarose (Sepharose 4B) substituted with 4-phenylbutylamine (PBA) or with ϵ-aminocaproyl- d-tryptophan methyl ester (ACTME) as compared to controls of untreated agarose and/or of cyanogen bromide treated agarose without addition of a substituting amine. 2. 2. At pH 8, α-chymotrypsin (3.4.4.5) and 7S γ-globulin bind virtually “irreversibly” to agarose-PBA, even in the presence of 1 m NaCl. Serum albumin, β-lactoglobulin, and ovalbumin also bind strongly to agarose-PBA at pH 8 and ⋍0.05 ionic strength but are to different extents eluted by 1 m NaCl. In each case elution by salt is enhanced by the presence of a polarity reducing agent, e.g., ethylene glycol. The findings suggest that binding of these proteins to agarose-PBA results from the combined (and possibly mutually reinforcing) effects of hydrophobic and electrostatic forces. 3. 3. Of the above proteins α-chymotrypsin and γ-globulin exhibit the 4. highest affinity for agarose-ACTME (as well as for agarose-PBA). Serum albumin displays strong affinity at pH 5. These proteins are to a large extent eluted from agarose-ACTME by 1 m NaCl, in contrast to the case of agarose-PBA. However, as with agarose-PBA, release from binding is greatly enhanced by the added presence of a polarity reducing agent. 5. 4. The applicability of hydrophobic affinity to protein separations is demonstrated by chromatography of a protein mixture on an agarose-PBA column. Furthermore, several esteroproteolytic enzymes in crude preparations were separated from the bulk of the protein and from each other by chromatography on agarose-ACTME.