One-pot synthesis of the stable CdZnTeS quantum dots for the rapid and sensitive detection of copper-activated enzyme.

Galactose oxidase is a copper-activated enzyme and have a vital role in metabolism of galactose. Much of the work is focused on determining the amount of galactose in the blood rather than measuring the amount of galactose oxidase to urge the galactosemia patients to restrict milk intake. Here, a simple and effective method was developed for Cu2+ and copper-activated enzyme detection based on homogenous alloyed CdZnTeS quantum dots (QDs). Meso- 2,3-dimercaptosuccinic acid (DMSA) was used as the reducing agent for preparing QDs and the highest quantum yield of CdZnTeS QDs was 69.4%. In addition, the as-prepared CdZnTeS QDs show superior fluorescence properties, such as good photo-/chemical stability. The DMSA was the surface ligand of the QDs, containing abundant -SH and -COOH, thus the surface ligands have a high affinity with Cu2+. Therefore, this developed probe can be applied for Cu2+ and galactose oxidase detection and shows a good sensitivity in the buffer. Then, this probe was successfully used for Cu2+ and galactose oxidase detection in real samples with the satisfactory results. The proposed fluorescence quenching strategy gives a new and simple insight for enzyme assay without the enzyme-catalyzed reaction.

Streptomyces coelicolor oxidase (SCO2837p): a new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2).

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.

Usefulness of galactose oxidase-Schiff test in rectal mucus for screening of colorectal malignancy.

Based on a "field-effect" theory in colon carcinogenesis, and the expression of the disaccharide tumor marker D galactose-beta-[1-->3]-N-acetyl-D-galactosamine (Gal-GalNAc) in the rectal mucus of patients with cancer and precancer of the colon, Shamsuddin developed a simple, accurate, inexpensive, easy to perform and rapid (< or = 15 min) screening test for colonic cancer and precancerous lesions. In this study we examined 137 rectal mucus samples of randomly selected patients with colorectal malignancy or other colorectal diseases to confirm the sensitivity and specificity of this test in Croatia. Additionally, to test the validity of the "field-effect" theory, that the mucosa away from the obvious cancer will show abnormalities as a result of the generalized effect of the carcinogen throughout the entire field of the target tissue, we also monitored a subset of 53 patients post-operatively. Individuals free of colonic or any other malignancies served as control (n = 31). The rectal mucin was smeared on membrane filter and developed by a sequential reaction of galactose oxidase (GO) and Schiff's reagent. The test results were correlated to the findings from colonoscopy/surgery and histopathology. The sensitivity of the test was shown to be 100% and the specificity was 96.8% (p < 0.001). Interestingly, the test was positive in 60% (32 of 53) of the samples collected from patients after tumor resection, showing the persistence of the biochemical changes even though malignant tumors were removed, hence supporting the field-effect phenomenon of carcinogenic stimuli. Five patients out of these 32 (16%) postoperative cases with positive GO test had a tumor recurrence within a year (0.05 < p < 0.10), suggesting that a persistently positive GO test in this population may serve as a predictor of tumor recurrence. We conclude that Gal-GalNAc is an early and intermediate biomarker, suitable not only for the detection of malignancy in its inception, but also for monitoring of people at high risk for cancer, and the efficacy of the cancer therapy as well as secondary prevention by this technology.

Production of mycotoxins by galactose oxidase producing "Fusarium" using different culture media

The original isolate of the galactose oxidase producing fungus "Dactylium dendroides, and other five galactose oxidase producing "Fusarium" isolates were cultivated in different media and conditions, in order to evaluate the production of 11 mycotoxins, wich are characteristic of the genus "Fusarium": moniliform, fisaric acid, deoxyvalenol, fusarenone-X, nivalenol, 3-acetyldeoxynivalenol, neosolaniol, zearakenol, zeralenone, acetyl T-2, and iso T-2. The toxicity of the culture extractes to "Artemia salina" larvae was tested

Gal-GalNAc: a biomarker of colon carcinogenesis.

The disaccharide tumor marker Gal-GalNAc visualized by galactose oxidase-Schiff sequence is commonly present in cancer cells and in rectal mucous of patients with colon cancer. The expression of this marker on tissue sections taken during experimental colon carcinogenesis shows excellent correlation with human precancerous lesions and even higher percentage of colon cancers express this marker, whereas, no expression is seen in the normal human large intestine. Multifocal expression of the marker is seen throughout the entire colon of patients with precancer and cancer; these include dysplasia, dilated and distorted crypts, regenerative dysplasia and hyperplastic crypts, as well as the morphologically normal crypts remote from cancer. Nearly identical pattern of Gal-GalNAc expression throughout the entire colon also appear during rat colon carcinogenesis induced by azoxymethane including non-expression by the normal and regenerative epithelia during wound healing following mechanical injury. Thus, Gal-GalNAc detected by the simple technique of galactose oxidase-Schiff sequence, is a biomarker that appears during the very early stages of progression of carcinogenesis. The expression pattern supports the field effect theory of carcinogenesis and also explains the basis for mass screening for cancer and precancerous conditions. Chemoprevention strategy using Gal-GalNAc as an intermediate marker detected by accurate and cost-effective rectal mucus test may have great potential.

Chemiluminescent detection for capillary electrophoresis and EMMA enzyme assays.

A large number of analytically and physiologically significant enzymes produce or consume hydrogen peroxide (H2O2). Post-column reaction of H2O2 with luminol in an alkaline solution results in chemiluminescence. A prototype reactor/detector for CE with chemiluminescent detection (CLD) of H2O2 was constructed and utilized. Two variations of the detection cell illustrated the difference between an analyte-limiting (stagnant-cell) and a window-limiting (swept-cell) detection scheme. The stagnant-cell detector had a low limit of detection (39 fmol at S/N = 3), but poor separation efficiency (1100 theoretical plates). The swept-cell detector demonstrated greater efficiency (226,000 theoretical plates), but a detection limit one order of magnitude higher (380 fmol). Several enzyme assays were developed using electrophoretically mediated microanalysis (EMMA) with chemiluminescent monitoring of H2O2. EMMA can be used to perform enzyme assays in a capillary format using electrophoretic mixing. Accumulated product of an incubation is swept to the detector and traditionally monitored by UV-VIS absorbance, fluorescence, or electrochemical detection. Higher sensitivity may also be obtained from the use of CLD. Chemical amplification with galactose oxidase and glucose oxidase resulted in the production of H2O2, while catalase enzymatically consumed H2O2. This work showed a CLD limit of 9300 molecules in the case of catalase.

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.

[A new screening method for colorectal cancer as a replacement for the hemoccult blood test].

A new screening test to detect a colorectal (CR) cancer (Shamsuddin, 1988) is reviewed. This test (Shams' test) is based on enzymatic detection of beta-D-Gal(1----3)-D-GalNAc (T-antigen) in the CR mucin. The sample and the reagent for this test are stable, the reaction process is simple, and the result is accurate and well reproducible. In contrast to the immunologically-detected fecal occult blood test, the sensitivity and specificity for CR cancers are surprisingly high, the percentage values in using the Shams test having been found to be 100% and 93.1%, respectively (Shamsuddin). In our pilot study using Japanese patients, the sensitivity was more than 80%. In view of these findings, the Shams' test can be considered a suitable replacement for the presently used immunological fecal occult blood test in screening CR cancers.

Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9).

Galactose oxidase from Dactylium dendroides was shown to contain one molecule of covalently bound pyrroloquinoline quinone (PQQ/enzyme molecule. From the spectroscopic characteristics reported for the enzyme forms, a mechanistic role for PQQ could be deduced. In analogy with other quinoproteins, the initial formation of a PQQ-substrate adduct is proposed. Following internal hydrogen transfer, leading to aldehyde product and reduced pyrroloquinoline quinone, reoxidation of the organic cofactor with molecular oxygen could be mediated by the PQQ-liganded copper ion with concomitant formation of hydrogen peroxide. With PQQ as an additional (two-electron) redox center the occurrence of a "superoxidized" enzyme form must be considered. Possible consequences of this view, in relation to a physiological function of the enzyme and interpretation of ESR data, are discussed.

Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.

Evaluation of an immobilized-enzyme analyzer for measuring galactose in serum.

Reportedly, galactose provides an alternative carbohydrate source and improved homeostatic regulation of glucose in the premature infant. Because of its potentially toxic effects, sensitive methods are needed for monitoring its concentration during therapy. We evaluated an immobilized galactose oxidase/hydrogen peroxide electrode system and a modified homogeneous enzymic method. Both methods are suitable for measuring galactose in a small sample and are comparably precise. The latter method gives superior analytical recoveries below 100 mg/L, but is linear in absorbance response to only 300 mg/L. We find the immobilized-enzyme method superior for monitoring treatment of neonates with galactose, because it requires only a few minutes and 25 microL of serum, and the analytical procedure is simpler.