An alternative coupling procedure for preparing activated sepharose for affinity chromatography of penicillinase.
Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axèn et al. (1967). Porath and Sunberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. alpha-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8-0-8-6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41% of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.
D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D004800:Enzymes, Immobilized; D005029:Ethylenediamines; D006863:Hydrogen-Ion Concentration; D008712:Methicillin; D010405:Penicillinase / Q000302:isolation & purification*; D012685:Sepharose; D013450:Sulfones
D004800:Enzymes, Immobilized; D005029:Ethylenediamines; D013450:Sulfones; D012685:Sepharose; D002918:Chymotrypsin; D010405:Penicillinase; D003488:Cyanogen Bromide; D008712:Methicillin