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.

Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

“…2A) C6-SANH on actin and C6-SFB [42], [43], [44] on a-rIgG circumvented the formation of extensive actin-actin or antibody-antibody cross-linking. The latter problems are seen when homobifunctional linkers such as glutaraldehyde [13] are used.…”

“…A key result is the realization of a novel approach for covalent antibody-attachment to cytoskeletal filaments using heterobifunctional cross-linkers [42], [43], [44]. Unlike the cross-linkers used previously for attachment of antibodies to microtubules [15], the heterobifunctional linkers allow attachment of both poly and monoclonal antibodies to the filaments.…”

Translational actomyosin research: fundamental insights and applications hand in hand

“…Risks of aggregate formation also exist if homobifunctional crosslinkers (e.g., glutaraldehyde) (Bachand et al 2006) are used to covalently immobilize antibodies to the cytoskeletal filaments. However, the more recent development of immobilization strategies, using heterobifunctional cross-linkers (Byeon et al 2010; Grotzky et al 2011; Iyer et al 2011), overcomes this obstacle, i.e., it prevents antibody–antibody or actin–actin cross-linking. A version of this approach, allowing attachment of polyclonal antibodies to microtubules has been studied (Carroll-Portillo et al 2009a) whereas a more versatile method that allows attachment of both monoclonal and polyclonal antibodies was applied quite recently to actin filaments (Kumar et al 2011, 2012, manuscript submitted).…”

“…Thus, the covalent antibody immobilization on actin filaments via heterobifunctional cross-linkers (Byeon et al 2010; Grotzky et al 2011; Iyer et al 2011) would allow attachment of antibodies to the actin filaments in solution thereby overcoming the limitation related to antibody attachment via biotin–streptavidin on the device surface. Further, by exploiting the high speed of HMM driven actin filaments and their low flexural rigidity (Vikhorev et al 2008a) we expect that actomyosin-driven concentration of analyte on a target (detector) zone should be possible in seconds which is faster than in recent high-sensitivity methods (Georganopoulou et al 2005; Nam et al 2003).…”

Chaperonin–Dendrimer Conjugates for siRNA Delivery

“…This linker chemistry has the advantage that the conjugation is stable in a wide pH range and that it can be quantified by UV–Vis spectroscopy 19. Cysteines (Cys) in the THS cavities were first reacted with the heterobifunctional linker maleimido trioxa‐6‐formylbenzamide (MTFB).…”