Preparation of Catalytically Active, Covalent alpha-Polylysine-Enzyme Conjugates via UV/Vis-Quantifiable Bis-aryl Hydrazone Bond Formation

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between alpha-poly-D-lysine (PDL) and either alpha-chymotrypsin (alpha-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 alpha-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 alpha-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.