Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
Biomacromolecules. 2011 Jan 10;12(1):134-44. doi: 10.1021/bm101074s. Epub 2010 Dec 20.
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.
研究了共价 UV/可见定量双芳基腙键的形成，用于制备α-聚-D-赖氨酸（PDL）与α-糜蛋白酶（α-CT）或辣根过氧化物酶（HRP）之间的缀合物。通过将连接试剂琥珀酰亚胺 6-肼基烟酸丙酮腙（S-HyNic，在 pH7.6）和琥珀酰亚胺 4-甲酰苯甲酸（S-4FB，在 pH7.2）分别修饰 PDL 和酶的游离氨基基团。然后在 pH4.7 下对修饰的 PDL 和酶进行缀合，由此获得携带几个酶的聚合物链。通过在 354nm 处分光光度法研究双芳基腙键形成的动力学。通过使用 N-琥珀酰亚胺-L-丙氨酸-L-丙氨酸-L-脯氨酸-L-苯丙氨酸对硝基苯胺（用于 α-CT）和 2,2'-偶氮双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（ABTS，用于 HRP）作为底物，确认了形成缀合物后酶活性的保留。因此，不仅可以温和高效地制备和方便地定量聚阳离子α-聚赖氨酸与酶的缀合物，而且还可以完全保留酶活性。
