Hydrophobic Affinity Chromatography of Proteins

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 E-aminocaproyl-n-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. At pH 8, cu-chymotrypsin (3.4.4.5) and 7s y-globulin bind virtually “irreversibly” to agarose-PBA, even in the presence of 1 M NaCl. Serum albumin, ,8-lactoglobulin. and ovalbumin also bind strongly to agarose-PBA at pH 8 and -0.05 ionic strength but are to different extents elutrd 1)~ 1 M NaCl. In each case elution by salt is enhanced by the presencr 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 rlrcl rost::tic forces. 3. Of t,he above proteins cY-chymotrypsin and y-globulin exhibit the highest affinity for agarose-ACTME (as well as for agarose-PBB). Serum albumin displays strong affinity at pH 5. These proteins are to a large extent eluted from agarose-ACTME by 1 ELI NaCl, in contrast to the KW of agarose-PBA. However. as with agarose-PBA, relcasc from binding is greatly enhanced by the added presence of a polarity reducing agent. 4. The applicability of hydrophobic affinity to protrin separations is demonstrated by chromatography of a protein mixture on an agarose-PBA column. Furthermore, several esteroproteolytic enzymes in crude prcparations were separated from the bulk of the protrin and from each other b> rhromatography on agarose-BCTME.