Application of a long-term enhanced xanthine oxidase-induced luminescence in solid-phase immunoassays

The use of xanthine oxidase (XO) as a label in immunoanalysis has not been previously reported. This can be explained by the difficulties encountered in XO assays (poor sensitivity and versatility) and the competitive inhibition of the enzyme by allopurinol, a widely used hypouricemic agent. We demonstrate here that both difficulties can be circumvented. (i) The XO-dependent luminescent signal related to the oxidation of luminol is dramatically enhanced in the presence of iron-EDTA complex and sodium perborate in alkaline buffer. The mechanism of this enhancement is consistent with an O2-driven Fenton reaction, leading to the production of highly reactive OH radical. (ii) Residual inhibition of solid-phase bound XO by serum allopurinol and its metabolites is spontaneously reversible and can be prevented by the presence of folic acid or azahypoxanthine in the incubation buffer. With these two problems solved, XO can be classified as a choice label in immunoanalysis with the following properties: (i) high detection sensitivity (3 amol label), (ii) long-term luminescent signal (several days), (iii) versatile preparation and stability of conjugates, and (iv) long-term stability of the luminescence reagent. As an example of application, some data concerning total IgE and direct 17 beta-estradiol assays are described. Several other luminescent immunoassays of large and small molecules have been developed using XO conjugates as tracer (free and total T4, ultrasensitive thyroid stimulating hormone, CA 19.9, prolactin, hCG, specific IgE, anti-toxoplasma, and anti-chlamydia IgG), thus proving that XO can be classified as a universal label.     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

Conjugation position of quercetin glucuronides and effect on biological activity

Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4′- > 3′- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The K_i for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4′- > 3′- > 7- > 3-, with quercetin-4′-glucuronide a particularly potent inhibitor (K_i = 0.25 μM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations.     

Characteristics and detection principles of a new enzyme label producing a long-term chemiluminescent signal

A new enzyme label system is described which is superior to all existing chemiluminescence labels used in immunoassays. The system consists of the enzyme xanthine oxidase with hypoxanthine as substrate. The signal reagent contains perborate, an Fe–EDTA complex and luminol. The enzyme preparation and the signal reagent are very stable upon storage. The main features of the system are a long duration of the chemiluminescent signal (half-life time of 30 hours) and a very low limit of detection (about 3 amol). Possibilities and implications for the use of various measuring system are discussed.     

Immobilized xanthine oxidase: Kinetics, (in)stability,and stabilization by coimmobilization with superoxide dismutase and catalase

Milk xanthine oxidase was immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to n-octylamine-substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (K_i′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the K_i for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (K_m′) for adsorbed xanthine oxidase was also higher than the K_m for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O₂^? and H₂O₂ side-products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde-crosslinked gelatin matrix, considerably enhanced the operational half-life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).     

Microwave assisted synthesis of naphthopyrans catalysed by silica supported fluoroboric acid as a new class of non purine xanthine oxidase inhibitors

A series of naphthopyrans was synthesized employing silica supported fluoroboric acid under solvent free conditions in a microwave reactor. The catalytic influence of HBF₄–SiO₂ was investigated in detail to optimize the reaction conditions. The synthesised compounds were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure–activity relationship analyses have also been presented. Among the synthesised compounds, NP-17, NP-19, NP-20, NP-23, NP-24, NP-25 and NP-26 were the active inhibitors with an IC₅₀ ranging from 4 to 17 μM. Compound NP-19 with a thiophenyl ring at position 1 emerged as the most potent xanthine oxidase inhibitor (IC₅₀ = 4 μM) in comparison to allopurinol (IC₅₀ = 11.10 μM) and febuxostat (IC₅₀ = 0.025 μM). The basis of significant inhibition of xanthine oxidase by NP-19 was rationalized by its molecular docking at MTE binding site of xanthine oxidase.     

Development of enzyme immunoassays for leukotrienes using acetylcholinesterase

We have developed sensitive solid phase enzyme immunoassays (EIA) to analyze quantitatively leukotrienes (LTs) using acetylcholinesterase from Electrophorus electricus as a label for LTB4, LTC4 and LTE4. However, because of problems specific to LTs, we used different coupling procedures to prepare LTs conjugates necessary for the production of antibodies and for the preparation of enzymatic tracers. For the immunogens, all LTs were coupled to bovine serum albumin using glutaraldehyde (ethylene diamine was used to add an amino group to LTB4). Immunizations in rabbits were done following classical procedures. For the enzymatic tracers, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was selected to conjugate the LTs via their amino groups to acetylcholinesterase. Titers of the different antisera ranged from 1:30,000 (LTE4), 1:40,000 (LTC4) to 1:50,000 (LTB4) and sensitivities (IC50) were 5.5 pg, 4.3 pg and 2.4 pg, respectively. Cross reactivities were also examined against other LTs. Sensitivities and specificities of the different systems were dependent on the conditions of incubation (temperature). Validation of the technique was done (i) after spiking known amounts of LTC4 in plasma and measuring the substance added after prior extraction and purification, (ii) by analyzing the supernatant of human neutrophils suspended in buffer or in plasma, (iii) by measuring LTE4 in urine. Due to the background provided by these complex matrixes, quantitation was performed after addition of [3H]LTs for recovery, protein precipitation, extraction by Sep-PakR and purification by HPLC. Measurement of LTs can be done in biological fluids with the same ease and advantages as other enzyme immunoassays that we have previously developed for eicosanoids analysis.     

Effect of delta sleep-inducing peptide on lipid peroxidation and xanthine oxidase activity in rat tissues during cold stress

I. p. administration of exogenous delta-sleep-inducing peptide (DSIP) decreased the amount of diene conjugates and Schiff bases in the liver and brain in rats. The xanthine oxidase activity, at that, did not change. Cold stress enhanced the xanthine oxidase activity well as the amount of diene conjugates and Schiff bases. Preliminary administration of the delta-sleep-inducing peptide to cold-exposed animals diminished the xanthine oxidase activity and lipid peroxidation in the liver and brain. Protective effects of the DSIP under stress is discussed.     

In silico and bio assay of juvenile hormone analogs as an insect growth regulator against Galleria mellonella (wax moth) – Part I

Juvenile hormone (JH) analogs are nowadays in use to control harmful pests. In order to develop new bioactive molecules as potential pesticides, we have incorporated different active structural features like sulfonamide, aromatic rings, amide group, and amino acid moiety to the base structure. We have screened a series of designed novel JH analogs against JH receptor protein (jhbpGm-2RCK) of Galleria mellonella in comparison to commercial insect growth regulators (IGRs) – Pyriproxyfen (T₁) and Fenoxycarb (T₂). All analogs exhibit the binding energy profile comparable to commercial IGRs. Based upon these results, a series of sulfonamide-based JHAs (T₃–T₈) as IGRs have been synthesized and characterized. Further, the efficacy of synthesized analogs (T₃–T₈) and commercial IGRs (Pyriproxyfen and Fenoxycarb) has been assessed against fourth instars larvae of G. mellonella under the laboratory conditions. LC₅₀ values of all the analogs (T₁–T₈) against the fourth instars larvae were 9.99, 10.12, 24.76, 30.73, 38.45, 34.15, 34.14, 19.48 ppm and the LC₉₀ 153.27, 131.69, 112.15, 191.46, 427.02, 167.13, 217.10, 172.00 ppm, respectively. Among these analogs, N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamide (T₈) and N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene sulfonamide (T₇) exhibited the good pest larval mortality at different exposure periods (in hours) and different concentrations (in ppm) in comparison to in use IGRs- T₁ and T₂. Bio assay results are supported by docking at higher concentration. The present investigation clearly exhibits that analog T₈ could serve as a potential IGR in comparison to in use IGRs (T₁ and T₂). The results are promising and provide new array of synthetic chemicals that may be utilized as IGRs.     

A comparative study of myoglobin stability in the presence of Hofmeister anions of ionic liquids and ionic salts

To reveal the impact of ionic liquids (ILs) on the stability of proteins, a series of ILs possessing same 1-butyl-3-methylimidazolium cation [Bmim]⁺ with a set of Hofmeister anions such as SCN⁻, HSO₄⁻, Cl⁻, Br⁻, CH₃COO⁻ and I⁻ were used and their effects on the myoglobin (Mb) structure and stability were studied. For the sake of comparison and also to explore the extent of the stabilization behavior of ILs toward Mb stability, we have chosen a set of ionic salts (I_s) of a fixed sodium cation (Na⁺) with the same series of anions such as SCN⁻, SO₄⁻², Cl⁻, Br⁻, CH₃COO⁻ and I⁻. UV–vis, fluorescence and circular dichroism (CD) spectroscopic techniques were used in order to investigate the stability behavior of Mb in ionic species (I_s and ILs). The results reveal that both I_s and ILs had a negative influence on the stability of Mb. Apparently, the flexibility in the native structure of Mb gradually increases with the increase in the concentration of I_s and ILs at pH 7.0. Therefore, a sharp decrease in the transition temperature (T_m) of the native Mb is observed in the presence of I_s and ILs.     

Luminescent immunoassay using isoluminol derivatives

Isoluminol derivatives (ID) were early employed in the preparation of tracers for immunoassay. Their wide use was mainly due to their high quantum efficiency, low molecular weight, well-known chemical structure, low cost and high stability. Moreover the light efficiency of some ID may be modified by specific binding to the antibody, thus allowing the development of homogenous immunoassays requiring no bound/free separation step. Some ID are commercially available under both the amino terminal and carboxyl terminal form for conjugation to respectively carboxy derivative of steroid and amino residues of protein. The linking reaction can be commonly carried on via active esters chemistry and can be easily accomplished within one day. Steroid conjugates can then be rapidly purified by silica gel thin layer chromatography, and protein conjugates by gel filtration on a short disposable column. In the field of steroid studies, isoluminol derivative conjugates were prepared for the immunoassay of almost all compounds of clinical interest. When dealing with protein, both antigens and antibody were labelled in this way, resulting in highly specific activity tracers for competitive and non-competitive immunoassays. Recently the labelling of streptavidin with amino-buty-ethyl-isoluminol allowed the development of very sensitive immunoassay methods which take advantage of the biotin-avidin system.     

Influenza virus H5N1 hemagglutinin (HA) T‐cell epitope conjugates: design,synthesis and immunogenicity

The influenza virus, major surface glycoprotein hemagglutinin (HA) is one of the principal targets for the development of protective immunity. Aiming at contributing to the development of a vaccine that remains the first choice for prophylactic intervention, a reconstituted model of HA, mimicking its antigenic properties was designed, synthesized and tested in mice for the induction of protective immunity. Four helper T lymphocyte [HTL (T₁, T₃, T₇ and T₈)] and four cytotoxic lymphocyte [CTL (T₂, T₄, T₅ and T₆)] epitopes were coupled in two copies each to an artificial carrier, SOC₄, which was formed by the repeating tripeptide Lys‐Aib‐Gly. The helical conformation of the SOC₄‐conjugates preserves the initial topology of the attached epitopes, which is critical for their immunogenic properties. Survival of immunized animals, ranged from 30 to 50%, points out the induction of protective immunity by using the SOC₄‐conjugates. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The mechanism of the activity-dependent luminescence of xanthine oxidase.

The weak luminescence that accompanies the aerobic xanthine oxidase reaction is inhibited by superoxide dismutase, by catalase, and by scavengers of hydroxyl radicals. It is also entirely dependent upon the presence of carbonate. It thus appears that the O₂ and H₂O₂ produced during the aerobic action of xanthine oxidase interact to generate OH which, in turn, reacts with carbonate to yield the carbonate radical (CO₃^?). The species that is directly responsible for light emission appears to be produced by a dimerization of carbonate radicals, since the light intensity was a function of the square of the carbonate concentration. The data provide no reason to suppose that the light-emitting species is singlet oxygen.     

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothioneen oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H₂O₂- and O₂⁻-dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn/metallothionein were preincubated with H₂O₂ and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein proteciion appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.     

Monofunctional derivatives of coproporphyrins for phosphorescent labeling of proteins and binding assays

p-Isothiocyanatophenyl derivatives of Pt(II)- and Pd(II)-coproporphyrin I are described as stable monofunctional reagents which enable simple covalent labeling of proteins and other biomolecules under mild conditions in aqueous solutions. Labeling procedure was optimized for antibodies, avidin, and neutravidin. Photophysical properties of resulting conjugates important for their use in binding assays based on time-resolved phosphorescence detection were studied. The functional activity and long-term storage stability of antibody conjugates were assessed in comparison with unmodified proteins. The new labels and their conjugates were evaluated in the solid-phase immunoassays using commercial time-resolved phosphorescence readers Victor(2) and Arcus-1230 (Wallac). Potential applications of these reagents in in vitro diagnostics are discussed.     

Preparation and characterization of monoclonal antibodies which recognise different gibberellin epitopes

The production and characterization of high-affinity monoclonal antibodies (McAb) to gibberellins (GAs) is reported. Hybrid myelomas were derived from immunisations with conjugates in which immunogenic proteins were linked to GA₁ at carbon-3 and to GA₄ and GA₉ at carbon-17. A series of McAb which display specificities allowing recognition of, and the discrimination between GA₁, GA₂₀, GA₄ and GA₉ is described. These McAb can be used in quantitative immunoassays for underivatised GAs.Abbreviations BSA bovine serum albumin - FCS foetal calf serum - GA_n gibberellin A_n - IFA incomplete Freund's adjuvant - KLH keyhole-limpet haemocyanin - McAb monoclonal antibody (bodies) - PBS phosphate-buffered saline     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

细菌黄嘌呤氧化酶的稳定性

研究pH、温度、金属离子和一些添加剂对黄嘌呤氧化酶稳定性的影响。结果表明：黄嘌呤氧化酶在pH4．5～7．5的范围内较稳定;反应最适温度为37℃。在常温25～35℃该酶比较稳定,经45℃处理2h．可保持50％左右,不同种类、不同浓度的金属离子对黄嘌呤氧化酶活性表现出程度不同的激活或抑制作用;添加谷氨酸和天门冬氨酸,能有效提高黄嘌呤氧化酶的存放稳定性.     

Ribonuclease Activity of the Peptides with Alternating Arginine and Leucine Residues Conjugated to Tetrathymidilate

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)₄G‐amide. Ribonuclease activity of the conjugates pep‐T₄ decreases in the order T₄‐(LR)₄G > T₄‐(LR)₂G > T₄‐(LLRR)2G > T₄‐(LR)₂PRLRG > S₂R₃‐Hmda‐T₄ ≥ R₅ ≠ (LR)₃. According to CD spectra, the free peptide (LR)₄G‐amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to α‐helix.