Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase

The measurement of oxalate in urine was improved by employing barley oxalate oxidase immobilized on alkylamine glass beads affixed in a glass beaker. The minimum detection limit was 3.6 mg l(-1) urine. The recovery of added oxalate was 88.9+/-9.2%. Within- and between-assay coefficients of variation (CV) were <4.0 and <9.4%, respectively. The urinary oxalate values were obtained by a commercial kit method and the present method showed a good correlation (0.999). The method is free from tedious handling of glass beads and Cl- interference.     

Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.     

An amperometric oxalate biosensor based on sorghum oxalate oxidase bound carboxylated multiwalled carbon nanotubes-polyaniline composite film

A highly sensitive, specific and rapid electrochemical oxalate biosensor was constructed by covalently immobilizing sorghum leaf oxalate oxidase on carboxylated multiwalled carbon nanotubes and conducting polymer, polyaniline nanocomposite film electrodeposited over the surface of platinum (Pt) wire using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry. The optimized oxalate biosensor showed linear response range of 8.4-272 μM with correlation coefficient of 0.93 and rapid response within 5 s at a potential of 0.4 V vs Ag/AgCl. The sensitivity was approximately 0.0113 μA/μM with a detection limit of 3.0 μM. Proposed oxalate biosensor was successfully applied to human urine sample.     

Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase

In order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M.F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance(s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5 +/- 0.8 microM (mean +/- S.D., n = 8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 microM, as estimated by the present method.     

Discrete analysis of bile acid in serum and bile with 3 alpha-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD+ during oxidation of bile acid by immobilized 3alpha-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in 0.065 M sodium phosphate buffer (pH 7.0). The minimum detection limit of the method was 4.8 pmol/L in the serum and 19.5 micromol/L in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were <1.0% and <0.2% in the serum and <0.2% and <0.6% in bile, respectively. A good correlation for bile acid in the serum (r1= 0.95) and in bile (r2 = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3alpha-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A).     

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

A method for co-immobilizing lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas spp., glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans and peroxidase from horseradish onto zirconia-coated alkylamine glass beads through glutaraldehyde coupling has been described. The co-immobilized enzymes retained 71.4% of initial specific activity with a conjugation yield of 43.6 mg/g support. The optimum pH and Km for triolein increased, while Vmax was decreased slightly, but incubation temperature for maximum activity remained unaltered after co-immobilization. The co-immobilized enzymes showed increased thermal and storage stabilities in cold, compared to their native form. Among the various metal salts tested, only CuSO4 caused inhibition of both free and co-immobilized enzymes. The co-immobilized enzymes showed better suitability over mixture of individually immobilized enzymes in determination of serum triglyceride.     

Indoleacetate oxidase activity of horseradish and other plant peroxidase isozymes

Zymographic analyses were carried out on a commercial peroxidasepreparation of horseradish (Cochlearia armorada) and crude extractsfrom several plant materials, such as roots of turnip, radish,spinach, sweet potato and morning glory, regarding the dualcatalytic activities of peroxidase- IA oxidase isozymes. A standardreacting mixture for the oxidase stain contained IA, TCP andFast Blue BB salt as substrate, promoting agent and dye coupler,respectively. An unidentified intermediate (or intermediates)resulting from enzymatic IA degradation was demonstrated tobe coupled with the diazonium salt and to form an insolublecomplex. At least four cathodal and four anodal peroxidase bandswere located in the horseradish preparation, the former appearingas dark IA oxidase bands and the latter as very faint bands. In the extracts from plant materials, less than half of thetotal peroxidase bands appeared as heavy to faint IA oxidasebands, whereas the remaining ones did not appear in the samecondition as that for the horseradish IA oxidase stain. Furthermore,it was noted that the intensity of each of the peroxidase bandswas not always correlated with that of the corresponding oxidasebands. ¹Contribution No. 683 from the National Institute of Genetics,Misima.     

Determination of serum triglycerides using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads

A method for determination of serum triglycerides (Tgs) using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling was developed and evaluated. The minimum detection limit of the method was 0.54 mM. The analytical recovery of added triolein in the serum was 97.55 +/- 1.5% (mean +/- S.D.). The mean value of serum Tgs, determined by the present method showed a good correlation (r = 0.984) with the Bayer's kit method, employing free enzymes. The within and between batch coefficients of variation (CV) were < 2.25% and < 1.35% respectively. No significant loss of activity was observed, when co-immobilized enzymes were reused for about 200 times and stored at 4 degrees C in distilled water. The cost of Tg determination for 200 serum samples was less, as compared with Bayer's kit method.     

The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.     

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.     

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.     

Degradation of oxalate in rats implanted with immobilized oxalate oxidase

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.     

Discrete analysis of serum uric acid with immobilized uricase and peroxidase.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.     

Homogeneous immunoenzyme analysis of human immunoglobulin G using horseradish peroxidase

The author studied the steady-state kinetics of cooxidation of 4-aminoantipyrine (AAP) with phenol and its derivatives--alpha-naphthol, o- and m-acetylaminophenols--by horseradish peroxidase and its conjugate with human immunoglobulins IgG (HRP-IgG). When phenol and AAP were used as peroxidase substrates, anti-human IgG antibodies stimulated the HPR-IgG enzyme activity in the presence of excess H2O2. A homogeneous enzyme immunoassay of human IgG was developed on the basis of this stimulating effect. The kinetics of the interactions between immunological reagents was studied. In the presence of 3% polyethylene glycol 6000, a complete antibody-antigen interaction proceeds at 37 degrees for 15-20 min. The sensitivity of the enzyme immunoassay is 350 ng/ml IgG, and the dynamic detection range is 0.35-15.0 mg/ml.     

Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

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.     

Softening of the packing density of horseradish peroxidase by a H-donor bound near the heme pocket

We use pressure tuning of spectral holes to estimate the compressibility of protein molecules by optical means. We found that the compressibility of mesoporphyrin-substituted horseradish peroxidase increases by a factor of three when it incorporates small aromatic H-donor molecules that bind in the vicinity of its heme pocket. Such a dramatic softening of its packing density corresponds to a jump from a compressibility range characteristic for the solid state into that characteristic for liquids.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.