PEG修饰的辣根过氧化物酶及其在非水介质中的性质

酶的化学修饰可以明显提高酶在有机相中的活力。通过氧化过氧化物酶（ＨＲＰ）的糖链后引入氨基再连接甲氧基聚乙醇（ＰＥＧ）５０００和在酶的肽链上连接ＰＥＧ５０００,发现ＨＲＰ多肽链上修饰后的酶在水相中的活力几乎没有变化,但通过氧化糖链连接ＰＥＧ的酶在水相中的活力下降近２倍。在甲苯及二氧六环含量较高的体系中,修和均呈上升趋势。特别在甲苯体系中两种修饰酶活力都比未经修饰的酶提高了近２倍。稳定性研究表明,不论     

Influence of heat treatment on rabbit liver ferritin

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Enhanced stability of β-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran

In order to enhance the stability of β-galactosidase, we conjugated the enzyme with dextran T-10 (M_r approx. 10 000). The conjugate contained 9–10 mol dextran/mol protein (β-galactosidase, M_r 68 000), and the specific activity retained after conjugation was 90 ± 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated β-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when β-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated β-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50°C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Fluid phase and mannose receptor-mediated uptake of horseradish peroxidase in mouse bone marrow-derived macrophages. Biochemical and ultrastructural study

A detailed study of horseradish peroxidase (HRP) uptake by in vitro cultured bone marrow-derived macrophages was undertaken. Biochemical quantitations performed over a wide range of HRP concentrations, in the absence or presence of yeast mannan, showed that these macrophages pinocytose HRP by both fluid phase and mannose receptor-mediated uptake. The relative contribution of these two types of endocytosis varied with the concentration of enzyme in the extracellular medium. A morphological study at the light and electron microscope levels conducted in parallel confirmed the biochemical data.     

A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity

Although various supports including nanomaterials have been widely utilized as platforms for enzymes immobilization in order to enhance their catalytic activities, most of immobilized enzymes exhibited reduced activities compared to free enzymes. In this study, for the first time, we used iron ions (Fe²⁺) and horseradish peroxidase (HRP) enzyme together to synthesize flowerlike hybrid nanostructures with greatly enhanced activity and stability and reported an explanation of the enhancements in both catalytic activity and stability. We demonstrated that Fe²⁺-HRP hybrid nanoflower (HNF) showed catalytic activity of ∼512% and ∼710%, respectively when stored at +4 °C and room temperature (RT = 20 °C) compared to free HRP. In addition, the HNF stored at +4 °C lost only 2.9% of its original activity within 30 days while the HNF stored at RT lost approximately 10% of its original activity. However, under the same conditions, free HRP enzymes stored at +4 °C and RT lost 68% and 91% of their activities, respectively. We claim that the drastic increases in activities of HNF are associated with to high local HRP concentration in nanoscale dimension, appropriate HRP conformation, less mass transfer limitations, and role of Fe²⁺ ion as an activator for HRP. Further biosensors studies based on enhanced activity and stability of HNF are currently underway.     

Purification and characterization of cholyl-CoA: taurine N-acetyltransferase from the liver of domestic fowl (Gallus gallus).

The enzymological basis for the ability of mammalian liver to conjugate bile acids with both glycine and taurine, and for non-mammalian liver to make only taurine conjugates, was investigated. The taurine-conjugating enzyme has been purified 1200-fold from the liver of domestic fowl and its properties compared with those of the glycine/taurine-conjugating enzyme from bovine liver [Czuba & Vessey (1980) J. Biol. Chem. 255, 5296-5299]. The enzyme from both species followed a Ping Pong mechanism. The enzymes were also similar with respect to their affinity for taurine, although the enzyme from domestic fowl would not bind glycine. The affinity of both for cholyl-CoA was quite similar, too, and both enzymes were inhibited reversibly by p-mercuribenzoate. The enzymes, however, were quite different in size. The enzyme from domestic fowl had a mol.wt. of 63000-65000 by both gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This is approx. 15 000 mol.wt. units larger than the enzyme from bovine liver, and suggests a loss of genome over the course of evolution as the basis for the altered specificity at the amino-acid binding site.     

Lectin-specific targeting of beta-glucocerebrosidase to different liver cells via glycosylated liposomes

Galactosylated and mannosylated liposomes were more efficient in transporting liposome-entrapped beta-glucocerebrosidase to liver compared to nonglycosylated liposomes. The enzyme entrapped to glycoside-bearing liposomes was found to be cleared at a much faster rate than that entrapped in liposomes having no sugar on their surface. Asialoorosomucoid and hydrolyzed mannan were found to inhibit both the clearance and the uptake of galactosylated and mannosylated liposomes, respectively, supporting involvement of lectin-sugar interaction. Further studies on the uptake of glucocerebrosidase by isolated liver cells revealed that the enzyme entrapped in mannosylated liposomes has much higher affinity for nonparenchymal cells whereas the assimilation of the entrapped enzyme into hepatocytes is clearly favored for liposomes having galactose on their surface.     

Preparation of catalytically active, covalent α-polylysine-enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between α-poly-d-lysine (PDL) and either α-chymotrypsin (α-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (for α-CT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic α-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.     

Enzyme activity assay for horseradish peroxidase encapsulated in peptide nanotubes

Encapsulation of horseradish peroxidase (HRP) inside a peptide nanotube (PNT) was demonstrated and its activity was measured. Enzyme assay verified that 0.16 μg of the enzymes were encapsulated in 1mg of PNTs. The encapsulation was also verified with TEM, UV-vis spectroscopy, and FTIR. The activity of the encapsulated HRP was examined for thermal stability, long-term storage stability, and resistance to a denaturant. They showed good storage stability, retaining its activity up to 90%, while the free HRP lost 50% of its activity over the course of 18 days. At 55 °C, the encapsulated HRP activity remained 20% higher than that of the free HRP. With the denaturant, guanidinium hydrochloride (GdmHCl), the encapsulated HRP activity was maintained around 10% higher than the free HRP. This result proves that the encapsulation of HRP inside the PNT may be an effective way to keep the enzyme activity stable in various environments.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

One-step screen-printed electrode modified in its bulk with HRP based on direct electron transfer for hydrogen peroxide detection in flow injection mode

The preparation and performances of screen-printed carbon electrodes modified in their bulk with HRP (HRP-SPCE) is reported. The resulting modified HRP-SPCE was prepared in a one-step procedure, and then was optimised as an amperometric biosensor operating at [0-100] mV versus Ag/AgCl in flow injection mode for hydrogen peroxide. The amperometric response was due to direct electron transfer (DET) between HRP and SPCE surface. Factors such as chemical modification of the enzyme or the nature and rate of the binder were investigated regards to their influence on the sensitivity, linear range and operational stability. The best performing HRP-SPCE in terms of sensitivity and operational stability was obtained when graphite powder was modified with HRP previously oxidised by periodate ion (IO(4)(-)).     

A biochemical and immunocytochemical study on the targeting of alglucerase in murine liver

Summary A current hypothesis is that functional glucocerebrosidase needs to be delivered to the lysosomes of tissue macrophages to guarantee successful enzyme therapy for Gaucher's disease. In this study, biochemical and immunohistochemical techniques were applied to identify in mice the localization of intravenously administered alglucerase (human modified placental glucocerebrosidase). Only in liver and spleen was a significant increase of glucocerebrosidase activity observed, with a maximum level at 15 minutes after enzyme infusion. The uptake of enzyme by liver was sufficiently high to allow more detailed studies on the (sub)cellular distribution of human alglucerase. The enzyme in liver is localized both in the endosomallysosomal system of the Kupffer cells and the endothelial cells lining the lumen of the sinusoids. Uptake by both of these types of cell is prevented by mannan. The results suggest that the cellular mechanisms responsible for improvement of Gaucher patients receiving alglucerase treatment is probably more complicated than previously recognized.     

Polarity in the transcytotic processing of apical and basal membrane-bound peroxidase-polylysine conjugates in MDCK cells.

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used to characterize a non-lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal-to-apical, but not apical-to-basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag-time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 microM) failed to inhibit this process, indicating that participation of the trans-Golgi network (TGN) in the trafficking of internalized conjugate is not the rate-determining step. Inhibition of HRP transport was found to be elicited by 50 micrograms/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface-bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal-to-apical, but not apical-to-basal, transport pathway; and this compartment can be exploited for the transcytosis of membrane-bound molecules.     

Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation

Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 degrees C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications.     

应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。