Peroxidase Activity of Myoglobin Variants Reconstituted with Artificial Cofactors.

Myoglobin (Mb) can react with hydrogen peroxide (H2 O2 ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and native mass spectrometry (nMS). The steady-state kinetics of guaiacol oxidation and 2,4,6-trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe-Ce6) and manganese chlorin e6 (Mn-Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild-type Mb, i. e. Fe-protoporphyrin IX-Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe-Ce6 into the mutant scaffold.

Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations.

The fight against tropical diseases such as malaria requires the development of innovative biosensing techniques. Diagnostics must be rapid and robust to ensure prompt case management and to avoid further transmission. The malaria biomarker hemozoin can catalyze atom transfer radical polymerizations (ATRP), which we exploit in a polymerization-amplified biosensing assay for hemozoin based on the precipitation polymerization of N-isopropyl acrylamide (NIPAAm). The reaction conditions are systematically investigated using synthetic hemozoin to gain fundamental understanding of the involved reactions and to greatly reduce the amplification time, while maintaining the sensitivity of the assay. The use of excess ascorbate allows oxygen to be consumed in situ but leads to the formation of reactive oxygen species and to the decomposition of the initiator 2-hydroxyethyl 2-bromoisobutyrate (HEBIB). Addition of sodium dodecyl sulfate (SDS) and pyruvate results in better differentiation between the blank and hemozoin-containing samples. Optimized reaction conditions (including reagents, pH, and temperature) reduce the amplification time from 37 ± 5 min to 3 ± 0.5 min while maintaining a low limit of detection of 1.06 ng mL-1. The short amplification time brings the precipitation polymerization assay a step closer to a point-of-care diagnostic device for malaria. Future efforts will be dedicated to the isolation of hemozoin from clinical samples.

Porphyrin-Cored Polymer Nanoparticles: Macromolecular Models for Heme Iron Coordination.

Porphyrin-cored polymer nanoparticles (PCPNs) were synthesized and characterized to investigate their utility as heme protein models. Created using collapsible heme-centered star polymers containing photodimerizable anthracene units, these systems afford model heme cofactors buried within hydrophobic, macromolecular environments. Spectroscopic interrogations demonstrate that PCPNs display redox and ligand-binding reactivity similar to that of native systems and thus are potential candidates for modeling biological heme iron coordination.

NMR spectroscopy of a single mammalian early stage embryo.

The resolving power, chemical sensitivity and non-invasive nature of NMR have made it an established technique for in vivo studies of large organisms both for research and clinical applications. NMR would clearly be beneficial for analysis of entities at the microscopic scale of about 1 nL (the nanoliter scale), typical of early development of mammalian embryos, microtissues and organoids: the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 µm) and sensitivity issues have prevented a widespread adoption of NMR. In this article we show how these limitations can be overcome to obtain NMR spectra of a mammalian embryo in its early stage. To achieve this we employ ultra-compact micro-chip technologies in combination with 3D-printed micro-structures. Such device is packaged for use as plug & play sensor and it shows sufficient sensitivity to resolve NMR signals from individual bovine pre-implantation embryos. The embryos in this study are obtained through In Vitro Fertilization (IVF) techniques, transported cryopreserved to the NMR laboratory, and measured shortly after thawing. In less than 1 h these spherical samples of just 130-190 µm produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectra vary from one sample to another despite their optical and morphological similarities, suggesting that the method can further develop into a non-invasive embryo assay for selection prior to embryo transfer.

NMR microsystem for label-free characterization of 3D nanoliter microtissues.

Performing chemical analysis at the nanoliter (nL) scale is of paramount importance for medicine, drug development, toxicology, and research. Despite the numerous methodologies available, a tool for obtaining chemical information non-invasively is still missing at this scale. Observer effects, sample destruction and complex preparatory procedures remain a necessary compromise. Among non-invasive spectroscopic techniques, one able to provide holistic and highly resolved chemical information in-vivo is nuclear magnetic resonance (NMR). For its renowned informative power and ability to foster discoveries and life-saving applications, efficient NMR at microscopic scales is highly sought after, but so far technical limitations could not match the stringent necessities of microbiology, such as biocompatible handling, ease of use, and high throughput. Here we introduce a novel microsystem, which combines CMOS technology with 3D microfabrication, enabling nL NMR as a platform tool for non-invasive spectroscopy of organoids, 3D cell cultures, and early stage embryos. In this study we show its application to microlivers models simulating non-alcoholic fatty liver disease, demonstrating detection of lipid metabolism dynamics in a time frame of 14 days based on 117 measurements of single 3D human liver microtissues.

Enzyme-initiated free radical polymerizations of vinyl monomers using horseradish peroxidase.

In this chapter, we highlight the use of horseradish peroxidase (HRP) as a catalyst to initiate free radical polymerizations of vinyl monomers under benign reaction conditions. A variety of vinyl monomers, including 4-acryloylmorpholine (AM), 2-hydroxyethyl methacrylate (HEMA), and poly(ethylene glycol) methyl ether acrylate (PEGA) were polymerized. The enzyme converts exogenous hydrogen peroxide into a usable radical source, which when coupled with a ß-diketone, yields a radical that initiates chain growth in the presence of monomers. The resulting polymers were characterized using nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). By using enzymatic free radical polymerizations, polymers can be generated in a sustainable, environmentally-friendly, and scalable fashion.

Biocatalytic ATRP in solution and on surfaces.

The promiscuity of enzymes allows for their implementation as catalysts for non-native chemical transformations. Utilizing the redox activity of metalloenzymes under activator regenerated by electron transfer (ARGET) ATRP conditions, well-controlled and defined polymers can be generated. In this chapter, we review bioATRP in solution and on surfaces and provide experimental protocols for hemoglobin-catalyzed ATRP and for surface-initiated biocatalytic ATRP. This chapter highlights the polymerization of acrylate and acrylamide monomers and provides detailed experimental protocols for the characterization of the polymers and of the polymer brushes.

Repurposing Biocatalysts to Control Radical Polymerizations.

Reversible-deactivation radical polymerizations (controlled radical polymerizations) have revolutionized and revitalized the field of polymer synthesis. While enzymes and other biologically derived catalysts have long been known to initiate free radical polymerizations, the ability of peroxidases, hemoglobin, laccases, enzyme-mimetics, chlorophylls, heme, red blood cells, bacteria, and other biocatalysts to control or initiate reversible-deactivation radical polymerizations has only been described recently. Here, the scope of biocatalytic atom transfer radical polymerizations (bioATRP), enzyme-initiated reversible addition-fragmentation chain transfer radical polymerizations (bioRAFT), biocatalytic organometallic-mediated radical polymerizations (bioOMRP), and biocatalytic reversible complexation mediated polymerizations (bioRCMP) is critically reviewed, and the potential of these reactions for the environmentally friendly synthesis of precision polymers, for the preparation of functional nanostructures, for the modification of surfaces, and for biosensing is discussed.