Development of the blood-brain barrier to horseradish peroxidase in the chick embryo

Summary The development of the adrenergic sympathetic innervation of the rabbit choroid plexus system was studied prenatally and up to two months after birth by a combination of fluorescence histochemistry (formaldehyde and glyoxylic acid methods) and quantitative enzymatic determinations of noradrenaline. The first signs of adrenergic nerves are found in the plexus of the third ventricle within the first day after birth. Fluorescent fibres subsequently appear in the choroid plexuses of the lateral ventricles (five days post partum) and the fourth ventricle (two weeks post partum). During the following development nerve fibres grow along blood vessels to form a plexus located between small vessels and the overlying epithelium. The nerve plexus, with varicose axon terminals, is fully developed at three weeks post partum, and maturation is then established by an increase in the number of terminals within the network of axons. There is a good agreement between (a) the development of the fluorescent nerves and histochemically visible adrenergic innervation, and (b) the tissue level of noradrenaline in the various choroid plexuses. Against the background of available information on the development of the secretory functions in choroid plexus, it is concluded that possibilities for a sympathetic neurogenic influence on the formation of cerebrospinal fluid exist already a few weeks after birth.     

Diffusion barrier to horseradish peroxidase in the vascular stria of the guinea pig

Summary Guinea pigs were given horseradish peroxidase intracardially and its diffusion in the vascular stria was studied. The Peroxidase spred freely among the intermediate cells and the marginal cells, but was never found to have passed zones occupied by tight junctions. It is concluded that the zones of tight junctions bordering the vascular stria represent a diffusion barrier to horseradish peroxidase.     

Stereospecificity of horseradish peroxidase

We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o-diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (delta1 and delta2). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower Km values for L- than for D-isomers. The kcat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same delta1 and delta2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates (K m) and in their transformation reaction rates (k cat).     

辣根过氧化物酶的热稳定剂

保持酶的天然状态和高催化特性具有重要的意义。本研究筛选了辣根过氧化物酶(HRP)的稳定剂并研究了其作用机制。结果发现硫酸镁和明胶能够显著提高HRP的热稳定性,并且两者具有协同作用。在硫酸镁和明胶组成的酶稳定剂存在的条件下,HRP在50oC保温80h后仍能保持89%的活性,常温下存放90d后可保持57%的活性,而未加稳定剂的对照样品中HRP的残留活性分别为6%和小于1%。通过对HRP的Soret带吸收光谱,色氨酸内源荧光,ANS荧光进行分析,揭示酶稳定剂可以明显降低在加热条件下HRP的变性程度,从而维持较为稳定的天然构象。     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Electrochemically deposited chitosan hydrogel for horseradish peroxidase immobilization through gold nanoparticles self-assembly

A new strategy for immobilization of horseradish peroxidase (HRP) has been presented by self-assembling gold nanoparticles on chitosan hydrogel modified Au electrode. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the deposited chitosan film with porous structure, which facilitates the assembly of gold nanoparticles and HRP. The resulting substrates were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The immobilized HRP displayed an excellent catalytic property to the reduction of H2O2 in the presence of methylene blue mediator. The resulting biosensor (HRP-modified electrode) showed a wide dynamic range of 8.0 microM-15 mM H2O2, and the linear ranges were 8.0 microM-0.12 mM and 0.50-12 mM, with a detection limit of 2.4 microM estimated at a signal-to-noise ratio of 3. Moreover, the biosensor remained about 85% of its original sensitivity after four weeks' storage.     

The demonstration of recurrent motor axon collaterals in the chick embryo by using horseradish peroxidase axonal transport]

Motoneurons were labelled by retrograde axonal transport of HRP applied to transected spinal nerves in 9-11-day chick embryos in the in vitro spinal cord preparation. Recurrent motor axon collaterals were revealed in 17 of 48 motor axons which could be followed in the edge regions of labelled motoneuronal pools. The results, coupled with author's earlier electrophysiological data, provide further evidence for the presence of the Renshaw inhibition in the avian spinal cord.     

Chemical stabilization of recombinant horseradish peroxidase

Summary Unglycosylated recombinant horseradish peroxidase (HRP C^*) had a half life of 21 minutes at 65°C compared with only 5 minutes for the plant enzyme (HRP). The half life of HRP C^* at 65°C increased by 5-fold following modification with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Tolerance to 60% 1,4-dioxan also increased whilst tolerance to 30% dimethylformamide was unchanged.     

Calcium-related properties of horseradish peroxidase.

Horseradish peroxidase has been shown to be a metalloprotein in which calcium contributes to the structural stability of the protein. Isoenzyme C and A contain 2.0 and 1.4 moles calcium/mole enzyme, respectively, which can be removed by treatment with guanidine hydrochloride and EDTA. Calcium-free isoenzyme C, but not isoenzyme A, reconstitutes upon addition of calcium and regains enzymatic activity. Free calcium readily exchanges with isoenzyme C, but only to a small extent with isoenzyme A. In addition the role of calcium in maintaining molecular conformation is evidenced by the effects of calcium removal from the isoenzyme C on the thermal stability of the protein.     

Immobilization of horseradish peroxidase onto kaolin

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer–Emmett–Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H₂O₂) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

Enhanced tissue adhesiveness of injectable gelatin hydrogels through dual catalytic activity of horseradish peroxidase

Development of bioadhesives with tunable mechanical strength, high adhesiveness, biocompatibility, and injectability is greatly desirable in all surgeries to replace or complement the sutures and staples. Herein, the dual catalytic activity of horseradish peroxidase is exploited to in situ form the hydroxyphenyl propionic acid‐gelatin/thiolated gelatin (GH/GS) adhesive hydrogels including two alternative crosslinks (phenol‐phenol and disulfide bonds) with fast gelation (few seconds – several minutes) and improved physicochemical properties. Their elastic moduli increase from 6.7 to 10.3 kPa by adding GS polymer that leads to the better stability of GH/GS hydrogels than GH ones. GH/GS adhesive strength is respectively 6.5‐fold and 15.8‐fold higher than GH‐only and fibrin glue that is due to additional disulfide linkages between hydrogels and tissues. Moreover, in vitro cell study with human dermal fibroblast showed the cell‐compatibility of GH/GS hydrogels. Taken together, GH/GS hydrogels can be considered as promising potential adhesive materials for various biomedical applications.     

Photooxidation of porphyrin in Mg-substituted horseradish peroxidase

Upon photoirradiation under aerobic conditions, the porphyrin prosthetic group in Mg-substituted horseradish peroxidase was oxidized to a mixture of its pi-cation radical and an oxidized product with an absorption band at 448 nm. The 448 nm compound was then converted to a 489 nm compound in the dark and the activation energy for the conversion was 19.3 kcal/mol. About 1 mol of O2 was consumed per mol of the 448 nm compound formed and no O2 consumption was seen in the dark reaction. The substitution of ethyl groups (meso) and hydroxyethyl groups (hemato) for the vinyl groups in protoporphyrin IX did not have an effect on the result. Under anaerobic conditions and in the presence of a suitable electron acceptor, the only photooxidation product of porphyrin was its pi-cation radical. The formation of hydroxyl radicals during irradiation under aerobic conditions was confirmed by the spin-trapping method. The formation of the above two radicals could be followed by ESR spectroscopy separately at a fixed magnetic field which was set to maximize each ESR signal. The rate of hydroxyl radical formation depended linearly on the concentration of Mg peroxidase. The photooxidation of porphyrin was slow and gave nonspecific product(s) when Mg protoporphyrin IX was present in the heme crevice of apomyoglobin or free in solution.     

Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothK _m andV _m in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.     

Unit cell dimensions of crystalline horseradish peroxidase

The preparation and unit cell dimensions of crystals of horseradish peroxide are described.     

DNA template-assisted modulation of horseradish peroxidase activity

The precise alignment of DNA molecules by Watson-Crick base-pairing combined with its polymeric characteristics have allowed DNA to be used as a template or scaffold for assembling materials. In this work, we investigate the role of calf-thymus DNA as a template for enhancing the horseradish peroxidase (HRP)-mediated oxidation of phenol and phenolic derivatives. The HRP-catalyzed oxidation of phenol into polyphenolic products and in presence of 4-aminoantipyrine into quinoneimine dye complexes is studied. Visible spectroscopy reveals an increased yield of both products of the enzymatic reaction in presence of calf-thymus DNA and is attributed to the prearrangement of the corresponding substrates on the DNA. The concentrations of calf-thymus DNA and the substrates are found to affect the nature of prearrangement and subsequent formation of polymeric or co-oxidation products. Also, phenolic derivatives with different aromatic substitutions display divergent propensities towards product formation in presence of the DNA template. Our results demonstrate the ability of calf-thymus DNA to modulate the activity of HRP and exercise control on the nature of products formed. This work highlights the potential of using DNA as a template for influencing enzymatic reactions involving aromatic substrates.     

Synthesis and properties of horseradish peroxidase copolymers

Conditions for copolymerization of native and sodium periodate-oxidized horseradish peroxidase (HTP; EC 1.11.1.7) have been optimized. Copolymerization products have been characterized electrophoretically, spectrally, and kinetically. Copolymers containing 2-3, 4, 5-7, and 9-10 molecules of the enzyme were found among the products of polymerization. The copolymers had lower values of D403/D280 than HRP. The copolymers had more ordered structures than the original HRP. Comparison of the thermal stability and kinetic characteristics of the fractions differing in the ratio of copolymers to the monomeric enzyme demonstrated that the polymeric products were more stable than HRP (in terms of resistance to high temperature or inhibitory effects of H202), but their kinetic activity was, on the whole, lower than that of the original enzyme.