Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.

Determination of serum glucose using co-immobilized glucose oxidase and peroxidase onto arylamine glass beads affixed on a plastic strip.

Glucose oxidase (GOD) from Aspergillus niger and horseradish peroxidase (POD) were co-immobilized onto arylamine glass beads affixed on a plastic strip with a conjugation yield of 28.2 mg/g and 43% retention of their initial specific activity. The coimmobilized enzymes showed maximum activity at pH 7.5 when incubated at 37 degrees C for 15 min. A simple, specific and sensitive method for discrete analysis of the serum glucose was developed employing this strip. The minimum detection limit of the method was 5 mg/dl. Within and between assay coefficient of variations for the serum were <5.6% and <10.6% (n = 6) respondely. A good correlation (r = 0.943) was found between the glucose values obtained by the enzyme colorimetric method employing free GOD and POD and the present method. The strip lost 50% of its initial activity after its 150 regular uses for a period of one month, when stored in reaction buffer at 4 degrees C. The method is cost-effective than the enzymic colorimetric method, as the enzyme strip is reusable.

Oxidation of phenols by horseradish peroxidase and lactoperoxidase compound II--kinetic considerations.

Oxidation of para substituted phenols by horseradish peroxidase compound II (HRP-II) and lactoperoxidase compound II (LPO-II) were studied using stopped flow technique. Apparent second order rate constants (kapp) of the reactions were determined. The kinetics of oxidation of phenols by HRP-II and LPO-II have been compared with the oxidation potentials of the substrates. Reorganization energies of electron-transfer of phenols to the enzymes were estimated from the variation of second order rate constants with the thermodynamic driving force.

The retinal distribution of ganglion cells with crossed and uncrossed projections and the visual field representation in the opossum

The retinal distribution of ganglion cells with crossed and uncrossed projections in the South American opossum, Didelphis marsupialis, was revealed by delivering HRP to one optic tract or to retinal targets of one hemisphere. The cells with uncrossed projections are restricted to the temporal retina, comprising 1/3 of the total retinal area, with a sharp transition at the naso-temporal boundary. Besides being distributed over the nasal 2/3 of the retina, cells with crossed projections are intermingled with those with uncrossed projections over the entire temporal retina. Quantitative analysis about the representation of the horizontal meridian on four specimens revealed that the maximun density of cells with uncrossed projection is on the average located at 3.2 mm (SD = 0.21), i.e. 34.8 deg, temporal to the optic disk, falling to 10% at 2.1 mm (SD = 0.14) or 22.8 deg. On the otherhand, the peak for cells with crossed projections is more nasally placed at 1.8 mm (SD=0.18), i.e. 19.6 deg. Between these two maxima, the site where in the densities of cells with crossed and uncrossed projections are about equal is on the average about 2.7 mm (SD = 0.25) form the optic disk, i.e. 29.3 deg. This estimate supports the hypothesis that the retinal itersection of the vertical meridian lies within the region of split representation of crossed and uncrossed ganglion cells. In addition, it was observed that the opossum's retina has a large contingent of cells with uncrossed projections temporal to an eccentricity of 2.7 mm from the optic disk, where it represents roughly 2/3 of the ganglion cells. These data corroborate the relevance of the opossum as a non-primate model for visual work

The neurogenesis of the callosal population of cortical cells in hamsters

The neurogenesis of the callosal subpopulation of cortical cells was determined in hamsters by associating incorporation of [3H]- thymidine injected on different embryonic days with horseradish peroxidase retrograde labelling in adulthood. Despite the great radial dispersion of migratory destinations of neurons born simultaneously, it was found that callosal birthdates in cortical area 6 extend from day E13 to day E15, a period that corresponds to the neurogenesis of layers III-V, where most callosal neurons come to be located in adults

Unusual distribution of ganglion cells in the retina of the three-toed sloth (Bradypus variegatus)

1. The distribution ans size of retinal ganglion cells labelled with horseradish preoxidase (HRP) were studied in flat-mounted retinas of three-toed sloths. 2. Massive injections of HRP solution were made throughout the thalamus and midbrain in anesthetized sloths in order to retrogradely label the retinal ganglion cell population. Twenty to thirty h later the eyes were and the retinas flat-mounted and reacted with phenylenediamine-HCL and H2O2 to label ganglion cells,thus distinguishing then from other cells int he same retinal layer. 3. Ganglion cell density graually increased from about 500 cell/mm2 at the far periphery to a peak of about 1,500 cells/mm2 in an area, termed the area centralis, deep in the inferior temporal retina. The presence of a vertical visual steak was also noted. 4. The area centralis contained a higher frequency of small ganglion cells than the peripheral retina where large cells preominated. 5. /the unusual postion of the area centralis and cisual streak in the retina can be explained by the slot's unique ability to rotate its head 180- while climbing upside-dow along horizontal branches so that the head is right-side-up. If it is assumed that the branch directly above the sloth's head needs to be visualized for accurate claw placement then the branch would be imaged on the inferior temporal retina in an area corresponding to the maximum density region

Enzymatic activation of the carcinogens 2 phenylethylhydrazine and 1,2 dimethylhydrazine to carbon centered radicals

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylgydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase-H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylgydrazine oxidation generates ten times carbon-centered radicals than 1,2-dimethylhidrazine oxidation. This results is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives