Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.     

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30 °C, which was shifted to 40 °C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40 °C after incubation for 1 h, respectively. The apparent kinetic constant values (K_ms) of wool-HRP were 10 mM for guiacol and 2.5 mM for H₂O₂, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.     

Removal of pentachlorophenol by immobilized horseradish peroxidase

Researches on the polymerization of aqueous pentachlorophenol (PCP) by the catalysis of horseradish peroxidase (HRP) with the existence of hydrogen peroxide (H₂O₂) were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of PCP and H₂O₂ that could influence the degradation were studied. Results showed that the optimum pH value for free enzyme was 5–6; relative higher temperature could accelerate the reaction greatly; PCP removal increased with an increase of enzyme concentration, and PCP (initial concentration 12.6 mg/L) removal percentage could reach nearly 70% under the highest enzyme concentration (about 0.05 u/ml) adopted in the experiment; removal percentage increased slightly with an increase of initial concentration of PCP, and when initial PCP concentrations were 13.0 and 0.7 mg/L, the removal percentages were about 73.7% and 35.7%, respectively; the molar ratio of the reaction between PCP and H₂O₂ was about 1:2.Based on the above results, researches on the removal of PCP by the immobilized HRP were conducted. The free HRP was immobilized on the polyacrylamide gel prepared by gamma-ray radiation method; then the immobilized HRP was filled into a column, and PCP was successfully removed by the immobilized HRP column. The results were compared with results using free HRP enzyme, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP, and when pH=5.15, the immobilized HRP could reduce PCP with initial concentration 13.4 mg/L to the concentration of 4.9 mg/L within 1 h, and the immobilized HRP column could be used to repeatedly.     

Ultrastructural study of the permeability of the guinea-pig placenta to horseradish peroxidase

Summary Horseradish peroxidase (HRP) was used to study macromolecule permeation into the guinea-pig placenta perfused in situ. When tissue culture medium 199 (TC 199) was used as fetal-side perfusate, the tracer reaction product was found only lining the fetal endothelium. When a longer period of perfusion with HRP in TC 199 was used, a small amount of reaction product was found in the subendothelial space and syncytiotrophoblastic vesicles, but not in maternal lacunae. In similar experiments using a Krebs bicarbonate Ringer (KRBG) as perfusate the tracer was found (i) lining the fetal endothelium, (ii) in the lateral intercellular spaces of the endothelium, (iii) in the subendothelial space, and (iv) in the maternal lacunae.It is therefore evident that the vehicle influenced the permeability of the guinea-pig placenta to horseradish peroxidase. As other studies have shown that perfusion of the fetal side with salt solution increases pore size, the results with TC 199 are regarded as more representative of the situation in the intact animal. It is therefore suggested that the fetal endothelium of the guinea-pig placenta may be largely impermeable to molecules of the size of horseradish peroxidase (4 nm) or larger.     

A combined horseradish peroxidase and Golgi study on the afferent connections of the ventrobasal complex of the thalamus in the cat

Summary Afferent connections to the ventrobasal complex (VB) of the thalamus were studied by means of retrograde transport of horseradish peroxidase (HRP) and by the Golgi-method. After HRP-injection into the VB, peroxidase-positive cells were observed contralaterally in the dorsal column nuclei (DCN), in the trigeminal nuclei and in the lateral cervical nucleus (LCN), and ipsilaterally in the somatosensory I (SI) and II (SII) cortical areas. Labeled cells of different shape and size were compared with neurons impregnated by the Golgi-technique. On the basis of HRP-labeling it is concluded that cells projecting to the VB are different in size and shape even within one region and that they correspond to the relay or efferent neurons observed in the Golgimaterial.     

Transport of horseradish peroxidase by processes of radial glia from the pial surface into the mouse brain

Summary The transport of horseradish peroxidase (HRP) applied to exposed pial surfaces of the brain was studied in newborn, 4-, 7- and 12-day-old, and adult mice. In the telencephalon the cell bodies of radial glia were found to accumulate the tracer. Labeled cells occurred in the subventricular zone of the lateral ventricle during the first postnatal week; they became gradually restricted to an area around the stria terminalis (ventrolateral ventricular corner) by day 12. At later stages no HRP transport could be traced from the surface of the telencephalon. In the cerebellum, HRP was transported from the surface to the cell bodies of Bergmann glia in all age groups studied including adult animals. It is concluded that radial glia and their derivatives share the capacity of transporting material between various cerebrospinal fluid compartments.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Effects of pH and pore characters of mesoporous silicas on horseradish peroxidase immobilization

Effects of immobilization pH and pore characters of mesoporous silicas (MPSs), MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; EC 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54 Å, while that of MCF with spherical cell and frame structure was 148 Å. Moreover, the MPSs synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and insignificant leaching were observed for all types of supports at pH 6. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respective immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41 < SBA-15 < MCF. HRP immobilized at pH 8 gave the highest storage stability (both at 4 °C and room temperature), and in opposition to pH 6. In addition, HRP immobilized in MCF was found to be the most stable under storage. The finding should be useful for the creation of biocatalysts and biosensors.     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

Impermeability of hagfish cerebral capillaries to horseradish peroxidase

Summary Brain capillaries and their permeability to intravenously injected horseradish peroxidase, HRP, (MW: 40,000) were examined electron-microscopically in an attempt to find a structural explanation for the poorly developed blood-brain barrier in the hagfish, Myxine glutinosa. In particular, it was the aim of this study to examine the role of the numerous endothelial vesicles and tubules in the transport of this tracer between blood and brain. Many of the vesicles and tubules were found to be in continuity with the luminal or abluminal surfaces, but tubules generating channels through the endothelial cells were never observed. The cleft between adjacent endothelial cells was obliterated by punctate junctions. HRP, which was allowed to circulate for up to 35 min, was not found in the basal lamina or in the surrounding brain parenchyma. Few of the luminal vesicles and tubules were marked by the tracer. In the intercellular cleft HRP was stopped by the junctions. It is concluded that the hagfish like other vertebrates has a blood-brain barrier to HRP, and the numerous vesicles and tubules occurring in hagfish brain endothelium are not involved in the transendothelial transport of this macromolecule.     

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V(m)/E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site.     

Immobilization of hemoglobin on chitosan films as mimetic peroxidase

Hemoglobin (Hb) was immobilized on the chitosan films using glutaraldehyde as a bifunctional agent. Atomic force microscopy (AFM) was used to examine the film surface in order to image the presence of Hb and Fourier transform infrared spectroscopy (FT-IR) was detected to elucidate the structural change of the immobilized Hb. The influences of several immobilization parameters were investigated, the optimum concentration of glutaraldehyde, pH and binding time were determined as 0.7%, 4.5 and 6 h, respectively. The enzymatic assay indicates that the immobilized Hb showed a higher thermal stability than that of free Hb, and the catalytic activity in organic solvents was also enhanced.     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

Preparation of photo-crosslinkable cinnamate modified hyaluronic acid for immobilization of horseradish peroxidase

In this work, a photo-responsive hydrogel membrane based on cinnamate-modified hyaluronic acid (HA-CM) was developed and safely cross-linked under UV light curing. The obtained material was effectively utilized for immobilization of horseradish peroxidase (HRP) enzyme via encapsulation and entrapment strategy with efficiency above 95%. The prepared HA-CM biopolymer was investigated before the UV curing using instrumental and spectral techniques including Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). During the UV irradiation, the progress of the cross-linking reaction was monitored by the UV–vis light spectroscopy. In addition, when the photo-induced cross-linking had accomplished, the morphological appearance of the hydrogel membrane was recorded using a scanning electron microscope (SEM). The HRP immobilized in HA-CM membranes displayed remarkable stability against the environmental pH changes especially under alkaline media and shift the optimum pH to 8 compared to the free HRP, which exhibited the highest activity at pH 7. Also, the entrapped enzyme was able to preserve above 85% of its catalytic activity at higher temperature values where the free enzyme had deactivated by approximately 50%. Moreover, HA-CM-HRP maintained 87% of its activity after 10 sequential reuse cycles, which indicate the economic value of the employed immobilization strategy.     

Resorption of horseradish peroxidase from the middle ear cavity of guinea pigs

Summary Horseradish peroxidase was injected into the middle ear and bulla of guinea pigs. In less than 5 minutes the peroxidase had reached the basement membrane, mainly through the epithelial intercellular spaces, and after 20 minutes it was observed in the lamina propria.     

Direct electrochemistry of horseradish peroxidase immobilized on DNA/electrodeposited zirconium dioxide modified,gold disk electrode

A novel H₂O₂ biosensor is described which is based on immobilization of horseradish peroxidase (HRP) on DNA/electrodeposited, ZrO₂/modified, gold electrode. The DNA is attached via its 5′ end to ZrO₂ and this provides a microenvironment for the immobilization of various biomolecules and promotes electron transfer between HRP and the electrode surface. Under optimized conditions, the biosensor reduced H₂O₂ linearly between 3.5 μM and 10 mM with a detection limit of 0.8 μM at a signal-to-noise ratio of 3. In addition, the developed biosensor shows an acceptable stability and repeatability. Importantly, the analytical methodology could be further developed for the immobilization of other proteins and biocompounds.