The use of horseradish peroxidase-lectin conjugates to monitor the purity of plasma-membrane preparations obtained from isolated cells.
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ABSTRACT:
SUBMITTER: Banga JP
PROVIDER: S-EPMC1163267 | biostudies-other | 1981 Aug
REPOSITORIES: biostudies-other
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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.
| S-EPMC3987668 | biostudies-literature
Project description:The present study explains computational methods to design thermostable horseradish peroxidase enzyme using the crystal structure available from Protein Data Bank (PDB ID: 6ATJ). Multiple mutations were introduced to the original enzyme and developed a model by using Modeler9.14. After designing the model functional effect was confirmed in terms of protein ligand binding by molecular docking using Autodock 4.2. The implementation of modeling steps is demonstrated in the context of performing mutations for particular amino acid residue on the ligand pocket of the horseradish peroxidase, to derive the desired ligand binding properties. The docking investigation of modelled HRP with Quercetindihydroxide using Autodock 4.2 software that six amino acid residues, P139, H42, A31, L174, A38, and G169 are involved in hydrogen bonding. More importantly, it provides insight into understanding and properly interpreting the data produced by these methods. The 3D model was docked with Quercetindihydroxide (a known horseradish modulator) to understand molecular interactions at the active site region.
| S-EPMC5322315 | biostudies-literature
Project description:When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.
| S-EPMC4116046 | biostudies-literature
Project description:Biotransformation of trans-resveratrol and synthetic (±)-?-viniferin in aqueous acetone using horseradish peroxidase and hydrogen peroxide as oxidants resulted in the isolation of two new resveratrol trimers (3 and 4), one new resveratrol derivative (5) with a dihydrobenzofuran skeleton, together with two known stilbene trimers (6 and 7), and six known stilbene dimers (8-13). Their structures and relative configurations were identified through spectral analysis and possible formation mechanisms were also discussed. Among these oligomers, trimers 6 and 7 were obtained for the first time through direct transformation from resveratrol. Results indicated that this reaction is suitable for the preparation of resveratrol oligomers with a complex structure.
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Project description:Hydrogels provide promising applications in tissue engineering and regenerative medicine, with silk fibroin (SF) offering biocompatibility, biodegradability and tunable mechanical properties. The molecular weight (MW) distribution of SF chains varies from ∼80 to 400 kDa depending on the extraction and purification process utilized to prepare the protein polymer. Here, we report a fundamental study on the effect of different silk degumming (extraction) time (DT) on biomaterial properties of enzymatically crosslinked hydrogels, including secondary structure, mechanical stiffness, in vitro degradation, swelling/contraction, optical transparency and cell behaviour. The results indicate that DT plays a crucial role in determining material properties of the hydrogel; decrease in DT increases β-sheet (crystal) formation and mechanical stiffness while decreasing degradation rate and optical transparency. The findings on the relationships between properties of silk hydrogels and DT should facilitate the more rational design of silk-based hydrogel biomaterials to match properties needed for diverse purpose in biomedical engineering.
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Project description:Ultrafast kinetic measurements of NO rebinding to horseradish peroxidase (HRP) are reported for the first time. The geminate kinetics are found to be exponential for all HRP samples studied. The ferric forms of HRP have NO geminate recombination time constants in the range of 15-30 ps, while the ferrous form has a time constant of approximately 7 ps. The simple exponential NO geminate kinetics found for HRP demonstrate that heme relaxation is not the underlying source of the nonexponential NO rebinding in myoglobin (Mb). The NO ligand escape rates from HRP are also determined, and they are found to depend dramatically on the presence or absence of the competitive inhibitor benzohydroxamic acid (BHA). The kinetic results indicate that, in contrast to Mb, there is direct solvent access to the distal heme pocket of HRP.
| S-EPMC2768277 | biostudies-literature
Project description:Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T(m) values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.
| S-EPMC1861803 | biostudies-literature
Project description:Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.
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Project description:A detailed mechanistic and kinetic study of enzymatically initiated RAFT polymerization is performed by combining enzymatic assays and polymerization kinetics analysis. Horseradish peroxidase (HRP) initiated RAFT polymerization of dimethylacrylamide (DMAm) was studied. This polymerization was controlled by 2-(propionic acid)ylethyl trithiocarbonate (PAETC) in the presence of H₂O₂ as a substrate and acetylacetone (ACAC) as a mediator. In general, well controlled polymers with narrow molecular weight distributions and good agreement between theoretical and measured molecular weights are consistently obtained by this method. Kinetic and enzymatic assay analyses show that HRP loading accelerates the reaction, with a critical concentration of ACAC needed to effectively generate polymerization initiating radicals. The PAETC RAFT agent is required to control the reaction, although the RAFT agent also has an inhibitory effect on enzymatic performance and polymerization. Interestingly, although H₂O₂ is the substrate for HRP there is an optimal concentration near 1 mM, under the conditions studies, with higher or lower concentrations leading to lower polymerization rates and poorer enzymatic activity. This is explained through a competition between the H₂O₂ acting as a substrate, but also an inhibitor of HRP at high concentrations.
| S-EPMC6403633 | biostudies-literature
Project description:The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75%, and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate of kBA ? (3.6 ps)(-1) is the dominant process, some CN(-) exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN(-) association rate, we find that the CN(-) escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ? (1-2) × 10(7) s(-1). This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1 × 10(7) s(-1)) under the same conditions. The analysis leads to an escape probability kout/(kout + kBA) ~ 10(-4), which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN(-) bimolecular association rate (170 M(-1) s(-1)), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN(-) bimolecular association rate is larger by ~10(3), making the CN(-) photolysis more difficult to observe.
| S-EPMC3631277 | biostudies-literature