Selective enzyme purification by affinity chromatography.

The purification of proteins by conventional procedures is frequently laborious and incomplete, and the yields are often low. Enzyme isolation based on a highly specific biological property-strong reversible association with specific substrates or inhibitors-has received only limited attention.‘-’ In affinity chromatography, the enzyme to be purified is passed through a column containing a cross-linked polymer or gel to which a specific competitive inhibitor of the enzyme has been covalently attached. All proteins without substantial afIinity for the bound inhibitor will pass directly t,hrough the column, whereas one that recognizes the inhibitor will be retarded in proportion to its affinity constant. Elution of the bound enzyme is readily achieved by changing such parameters as salt concentration or pH, or by addition of a competitive inhibitor in solution. The successful application of the method requires tl&t the adsorbent have a number of favorable characteristics. Thus, the unsubstituted matrix or gel should show minimal interaction with proteins in general, both before and after coupling to the specific binding group. It must form a loose, porous network that permits easy entry and exit of macromolecules and which retains favorable flow properties during use. The chemical structure of the supporting material must permit the convenient and extensive attachment of the specific ligand under relatively mild conditions, and through chemical bonds that are stable to the conditions of adsorption and elution. Finally, the inhibitor groups critical in the interaction must be sufficiently distant from the solid matrix to minimize steric interference with the binding processes. In this report the general principles and potential application of affinity chromatography are illustrated by results of its application to the purification of staphylococcal nucleaee, cr-chymotrypsin, and carboxypeptidase A. The solid matrix used in these studies wassepharose (a “beaded” form of the cross-linked dextran of highly porous structure, agaroses) which displays virtually all the desirable features listed above. Activation of the Sepharose by treatment with cyanogen bromidd* I results in a derivative that can be readily coupled to unprotonated amino groups of an inhibitory analog. The resultant Sepharose-inhibitor gel is a highly stable structure which has nearly ideal properties for selective column chromatography. Ezpesimenti Procedure.-M&tic&: Sepharose 4B wm obtained from Pharmacia, cyanogen bromide from Eastman, pdTp and benzoyl-Ltyrosine ethyl ester from Calbiochem. Staphylococcal nuclease (Foggi strain) was obtained by modification* of techniques described by Fuchs et al.# The following purified enzymea were purchased from Worthington: a-chymotrypsin (CDS 7LC); a-chymotrypsin, DFP-treated (CD-DIP 204); chymotrypsinogen A (CCC SCC) ; subtilisin VIII (MB 3080) ; trypsin, DFP-treated