title: Preparation of catalytically active, covalent α-polylysine-enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation.
abstract: Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between α-poly-d-lysine (PDL) and either α-chymotrypsin (α-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (for α-CT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic α-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.
authors.affiliation: Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Grotzky
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Manaka
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kojima
authors.forename: Peter
authors.initials: P
authors.lastname: Walde
mesh_terms: D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010777:Photochemistry / Q000379:methods; D011107:Polylysine / Q000737:chemistry*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011107:Polylysine; D006735:Horseradish Peroxidase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin