In Vitro Slow Fluctuation in Horseradish Peroxidase Activity

In vitro slow fluctuations in the level of horseradish peroxidase activity were observed in long-range experiments (72-144 h). Besides random fluctuations, regular slow oscillatory patterns with period lengths ranging from 10.0 to 39.0 h were detected by statistical analysis. The possibility that these oscillations in enzyme activity could have reflected changes in the physical environment of the experimental setup has been thoroughly examined and ruled out. Periodic exposition of the enzyme solution, otherwise kept in darkness, to blue light illumination was shown to influence the period of the oscillations. The changes in enzyme activity were correlated with a modification of the Michaelis constant estimated using guaiacol as substrate. This result was confirmed by the action of chemical modifiers of the enzyme, such as ferulic acid and rutin. It is thought that the observed oscillations in horseradish peroxidase activity are due to spontaneous and specific changes in the tridimensional structure of the enzyme in the thermic reservoir.     

Anomalous oxidation of MDL 73,404 by horseradish peroxidase

3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program "KINETIC 3.1" for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2'-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

Menadione-linked α-glycerophosphate dehydrogenase activity of motoneurons in rat soleus and extensor digitorum longus neuron pools

After injection of horseradish peroxidase into the soleus (slow twitch) and extensor digitorum longus (fast twitch) muscles, glycolytic enzyme activity as reflected by -glycerophosphate dehydrogenase activity of labeled motoneurons in the neuron pool was examined. No differences were found in glycolytic enzyme activity of motoneurons between slow twitch and fast twitch neuron pools.     

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.     

Relationship between transmural potential difference and smooth muscle slow waves and contractility in the rabbit small intestine in vitro

The relationship between transmural potential difference (PD) and smooth muscle electrical and mechanical activity was investigated in the rabbit ileum in vitro. Transmural PD was monitored using agar salt bridge electrodes connected via calomel half cells to an electrometer. Force displacement transducers recorded predominantly longitudinal smooth muscle activity. Concurrently, predominantly circular muscle activity was recorded at three sites using intraluminal pressure probes. At the same sites, suction electrodes monitored electrical activity of the smooth muscle. In all experiments, fluctuations in transmural PD were temporally linked to smooth muscle mechanical and electrical activity. The frequency of PD oscillations, electrical slow waves, and cyclic pressure changes were identical within each segment. Adrenaline abolished smooth muscle electrical spiking, all mechanical activity, and transmural fluctuations in PD. However, the slow waves were not abolished, though their frequency was increased. Phentolamine but not propranolol reversed the effects of adrenaline, thus slow wave frequency is influenced by alpha-adrenergic stimulation in the rabbit ileum. In conclusion, oscillations in transmural PD are unrelated to the ionic processes associated with the slow wave. However, they are in some way linked to smooth muscle contractile activity, possibly via an intrinsic neural mechanism as observed in the guinea pig.     

Oxidoreductase expression and protein storage in winter and spring cereals under extremal temperature condition

Cultivation of wheat and barley plants under temperature stress fluctuations was the reason of reduction of acid protein common storage in the tissues more than 25% and the reason of reduction of peroxidase and superoxide dismutase common activities. There were no changes of common phenoloxidase activity. Activity redistribution between separate fractions of enzyme spectra were linked with enzyme expression changes. Alteration of peroxidase/oxidase ratio for separate fractions of electrophoretical spectra has been defined as a result of extreme temperature influence over a period of plant growing. At the same time there were no alterations in peroxidase/superoxide reductase fraction ratio in seedlings. The Vrn1-loci did not have a single meaning for expression of investigated oxidoreductases because their influence was determined by the "organism-environment" definite situation. In "normal" growth circumstances there was no correlation between the type of development and qualitative and quantitative characteristics of oxidoreductase spectra. The alteration of definite enzyme izoform degree in forming of plant stress response was different for spring and winter genotypes that testifies dependence of the functional state of the probed enzymes on allelic composition of locus Vrn1.     

The dual oxygenase and peroxidase activities of porphobilinogen oxygenase and horseradish peroxidase: a study using the reaction with phenylhydrazine.

Porphobilinogen oxygenase and horseradish peroxidase show dual oxygenase and peroxidase activities. By treating porphobilinogen oxygenase with phenylhydrazine in the presence of H2O2 both activities were inhibited. When horseradish peroxidase was treated in the same manner only the peroxidase activity was lost while its oxygenase activity toward porphobilinogen remained unchanged. The phenylhydrazine treatment alkylated the prosthetic heme group of porphobilinogen oxygenase and N-phenylheme as well as N-phenylprotoporphyrin IX were isolated from the treated hemoprotein. In horseradish peroxidase the modified heme was mainly 8-hydroxymethylheme. The apoproteins of the alkylated enzymes were isolated and recombined with hemin IX. The oxygenase and peroxidase activities of porphobilinogen oxygenase were entirely recovered in the reconstituted enzyme, while the reconstituted horseradish peroxidase regained 75% of its peroxidase activity.     

Purification and characterization of chorion peroxidase from Aedes aegypti eggs

Previous study has shown that a peroxidase is present in the mature eggs of Aedes aegypti mosquitoes, and the enzyme is involved in the formation of a rigid and insoluble chorion by catalyzing chorion protein crosslinking through dityrosine formation. In this study, chorion peroxidase was solubilized from egg chorion by 1% SDS and 2 M urea and purified by various chromatographic techniques. The enzyme has a relative molecular mass of 63,000 as estimated by SDS-PAGE. Spectral analysis of the enzyme revealed the presence of the Soret band with a lambda(max) at 415 nm, indicating that chorion peroxidase is a hemoprotein. Treatment of the native enzyme with H2O2 in excess in the absence of reducing agents shifted the Soret band from 415 to 422 nm, and reduction of the native enzyme with sodium hydrosulfite under anaerobic conditions changed the Soret band from 415 to 446 nm. These results show that the chorion peroxidase behaves similarly to other peroxidases under oxidative and reductive conditions, respectively. Compared to other peroxidases, the chorion peroxidase, however, is extremely resistant to denaturing agents, such as SDS and organic solvents. For example, chorion peroxidase remained active for several weeks in 1% SDS, while horseradish peroxidase irreversibly lost all its activity in 2 h under the same conditions. Comparative analysis between mosquito chorion peroxidase and horseradish peroxidase showed that the specific activity of chorion peroxidase to tyrosine was at least 100 times greater than that of horseradish peroxidase to tyrosine. Chorion peroxidase is also capable of catalyzing polypeptide and chorion protein crosslinking through dityrosine formation during in vitro assays. Our data suggest that the characteristics of the chorion peroxidase in mosquitoes closely reflect its functions in chorion formation and hardening.     

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。     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Chemical modification of epsilon-amino lysine groups in horseradish peroxidase. Its effect on catalytic properties and spatial structure of the enzyme]

Effect of chemical modification of horseradish peroxidase lysine epsilon-amino groups by propionic, butyric, valeric, succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) on catalytic properties of the enzyme is investigated. All the preparations of modified peroxidase have 100% peroxidase activity for o-dianizidine at pH 7.0, which indicates the absence of lysine epsilon-amino group in the enzyme active site. pH-dependencies of modified peroxidase relative activity are studied; modification by anhydrides of monobasic acids is not found to result in changes of the relative activity pH-profile, while modification by succinic anhydride widens it. Absorption and circular dichoism spectra of native and modified peroxidase within 260--270 nm are obtained, some changes in the enzyme tertiary structure after its epsilon-amino groups modification are observed. Modification of four epsilon-amino groups by buturic and succinic anhydrides and of three epsilon-amino groups by TNBS is found to increase the regidity of protein surrounding of heme, and modification of six epsilon-amino groups by TNBS results in more unwrapped enzyme structure as compared with its native molecule.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

THE INTERACTION OF CATIONIC LIPOSOMES CONTAINING ENTRAPPED HORSERADISH PEROXIDASE WITH CELLS IN CULTURE

Cationic liposomes composed of sphingomyelin, cholesterol, and stearylamine were prepared with horseradish peroxidase trapped inside. Stable particles were formed in which 10–12% of the enzymic activity appeared to be located at, or near, the outer surface of the liposome. Adsorption and uptake of liposomes by HeLa cells were followed cytochemically by electron microscopy and quantitated by enzyme assay and by the distribution and fate of particles labeled with [¹⁴C]cholesterol and [¹²⁵I]horseradish peroxidase. The particles were adsorbed by HeLa cells at least 300 times as efficiently as was free horseradish peroxidase. Many of the particles remained at the cell surface, but numerous membrane-bound cytoplasmic inclusions were observed to contain peroxidase-staining material. In addition, many areas of the cell membrane gave a positive staining reaction. It was concluded that many particles (presumably the larger ones) did not gain access to the interior of the cells, many were phagocytized, and some enzyme was transferred to the cell membrane, perhaps as a result of fusion of the liposomal membrane with the cell membrane.     

Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Synchronous reversible alterations in enzymatic activity (conformational fluctuations) in actomyosin and creatine kinase preparations.

The phenomenon of synchronism of oscillations of actomyosin and creatine kinase activity in the whole volume of the enzyme preparations was analysed. The synchronous "conformational oscillations" were observed in concentrated gels of actomyosin and in diluted actomyosin and creatine kinase solutions (ATP-creatine N-phosphotransferase, EC 2.7.3.2). The macromolecules of proteins studied may be in two or four conformational states differing enzymatic activity. Large fluctuations become possible in a range of conditions wherein two or four different states, or conformers, are equiprobable. The synchronization of conformational changes of separate macromolecules is maintained with energy derived, for instance, from some oxidative process or dilution of the solution, the process being displayed as conformational oscillations.