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Adsorption of Horseradish Peroxidase on Metallic Nanoparticles: Effects on Reactive Oxygen Species Detection Using 2',7'-Dichlorofluorescin Diacetate.

ABSTRACT: The fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study acellular reactive oxygen species (ROS) production from nanoparticles (NPs). This study examined whether HRP adsorbs onto NPs of Mn, Ni, and Cu and if this surface process influences the extent of metal release and hence the ROS production measurements using the DCFH assay in phosphate buffered saline (PBS), saline, or Dulbecco's modified Eagle's medium (DMEM). Adsorption of HRP was evident onto all NPs and conditions, except for Mn NPs in PBS. The presence of HRP resulted in an increased release of copper from the Cu NPs in PBS and reduced levels of nickel from the Ni NPs in saline. Both metal ions in solution and the adsorption of HRP onto the NPs can change the activity of HRP and thus influence the ROS results. The effect of HRP on the NP reactivity was shown to be solution chemistry dependent. Most notable was the evident affinity/adsorption of phosphate toward the metal NPs, followed by a reduced adsorption of HRP, the concomitant reduction in released manganese from the Mn NPs, and increased levels of released metals from the Cu NPs in PBS. Minor effects were observed for the Ni NPs. The solution pH should be monitored since the release of metals can change the solution pH and the activity of HRP is known to be pH-dependent. It is furthermore essential that solution pH adjustments are made following the addition of NaOH during diacetyl removal of DCFH-DA. Even though not observed for the given exposure conditions of this study, released metal ions could possibly induce agglomeration or partial denaturation of HRP, which in turn could result in steric hindrance for H₂O₂ to reach the active site of HRP. This study further emphasizes the influence of HRP on the background kinetics, its solution dependence, and effects on measured ROS signals. Different ways of correcting for the background are highlighted, as this can result in different interpretations of generated results. The results show that adsorption of HRP onto the metal NPs influenced the extent of metal release and may, depending on the investigated system, result in either under- or overestimated ROS signals if used together with the DCFH assay. HRP should hence be used with caution when measuring ROS in the presence of reactive metallic NPs.

SUBMITTER: Kessler A

PROVIDER: S-EPMC8220500 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Dataset Information

Adsorption of Horseradish Peroxidase on Metallic Nanoparticles: Effects on Reactive Oxygen Species Detection Using 2',7'-Dichlorofluorescin Diacetate.

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