Electrochemically and Catalytically Active Reconstituted Horseradish Peroxidase with Ferrocene-Modified Hemin and an Artificial Binding Site

Abstract

A procedure for modification of hemin chloride by FcCH 2 NH 2 (Fc ferrocenyl) in the presence of 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide affords two main products 1 and 2 with mono-and bis-amidated propionic acid residues. Monoamidated conjugate 1 was loaded into the apoenzyme of horseradish peroxidase (HRP) to afford an electrochemically and catalytically active reconstituted enzyme Fc-HRP with remarkably altered substrate specificity. With ABTS as substrate, the reactivity of Fc-HRP drops threefold compared with native HRP as a result of a lowering of the maximal rate V m . Compared with HRP the reactivity of Fc-HRP towards water-soluble ferrocenes is even higher at low concentrations of the latter, the rate increase being accompanied by a change in rate law: in contrast to first-order kinetics in ferrocenes for native HRP, there is a Michaelis dependence for Fc-HRP. Molecular modeling suggests creation of an artificial hydrophobic binding site within a triangle confined by the ferrocenyl residue and the two phenyl rings of Phe 68 and 179. The site is believed to be responsible for the kinetically meaningful binding between ferrocene substrates and Fc-HRP which manifests in the saturation kinetics.

Influence of Macromolecular Crowding on the Oxidation of ABTS by Hydrogen Peroxide Catalyzed by HRP

“…The oxidation of ABTS by H 2 O 2 is such a process. The values of Michaelis-Menten parameters, ‫ݒ‬ ௫ and ‫ܭ‬ reported in diluted solution and the values that we obtained using similar conditions are comparable [39].…”

An Electrochemical Biosensor with Dual Signal Outputs: Toward Simultaneous Quantification of pH and O2 in the Brain upon Ischemia and in a Tumor during Cancer Starvation Therapy

“…Furthermore,astrong band at 1700 cm À1 observed in the IR spectrum of hemin chloride shifts to 1641 cm À1 in that of Hemin-Fc ( Figure S2, Supporting Information), which was also evidence that the carboxylic group in hemin chloride was successfully converted into an amide group (Scheme 1a). [8] Multi-walled carbon nanotube (MWNT) fibers were dry-spun from spinnable MWNT arrays at ar otating rate of 2000 rpm, which were synthesized by chemical vapor deposition in advance. [9] Thea s-prepared MWCNTs are straight and less constrained from the environment ( Figure 1a).…”

Apoenzyme Reconstitution as a Chemical Tool for Structural Enzymology and Biotechnology

“…[156,157] In an attempt to design an electrochemically active enzyme, Ryabov et al prepared heme-ferrocene conjugate 30 (Figure 11), in which aminederivatized ferrocene groups were covalently coupled to the propionate chains on the heme. [158] It was known from previous work that HRP reconstituted with heme modified with a monomethyl ester displays only about 20 % of the activity of native HRP. [119] Thus, it was questionable whether bulky substituents in these positions of the reconstituted heme enzymes would lead to functional enzymes.…”