四甲基联苯胺显色光度法测定过氧化氢酶活性

基于Fe2+-H2 O2-四甲基联苯胺(tetramethylbenzidine,TMB)显色体系测定过氧化氢酶(CAT)活性.在Fe2+催化作用下过氧化氢与四甲基联苯胺发生显色反应.加入CAT后,A值减小,显然CAT对显色反应具有阻抑作用.在优化实验条件下,CAT活性在0.00～0.10 U·mL-1之间与△A呈现良...     

Flow-injection assay of catalase activity.

A novel flow-injection assay (FIA) system with a double line for catalase activity was constructed in which an oxidase is immobilized and the substrate is continuously pumped to reduce the dissolved oxygen and to generate a given level of hydrogen peroxide. The catalase in a sample decomposed the hydrogen peroxide, and thus the increase in dissolved oxygen dependent on the activity was amperometrically monitored using a Clark-type oxygen electrode. Among the examined several oxidases, uricase was most suitable for the continuous formation of hydrogen peroxide from a consideration of the stability and the conversion efficiency. Under the optimum conditions, a linear calibration curve was obtained in the range from 21 to 210 units/mg and the reproducibility (CV) was better than 2% by 35 successive determinations of 210 units/ml catalase preparation. The sampling frequency was about 15 samples/h. The present FIA system was applicable to monitor the inactivation of catalase by glycation.     

Simple isocratic high-performance liquid chromatographic method for measuring pyridoxine kinase activity in crude biological extracts

To measure the enzymatic activity of pyridoxine kinase (EC 2.7.1.35) when pyridoxine or pyridoxamine are the substrates an additional enzymatic step is usually required. The products of the kinase activity, pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate, are oxidized, enzymatically, to pyridoxal 5'-phosphate (co-enzyme) which is then measured either spectrophotometrically or using apo-enzymes. In this report the enzymatic activity of pyridoxine kinase, in crude biological extracts, is assayed by a simple high-performance liquid chromatographic method which determines the amount of pyridoxine 5'-phosphate formed when pyridoxine is the substrate. The same method could be used when pyridoxamine is the substrate.     

Improved DPPH determination for antioxidant activity spectrophotometric assay

An improved procedure for determination of the residual DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical concentration was proposed taking into account the absorbance of both DPPH free radicals and DPPH nonradical (1,1-diphenyl-2-picrylhydrazine) stable form. The calculated residual DPPH free radical concentrations were compared with those obtained from a calibration curve and variation coefficients below 10 % were found.     

Different spectrophotometric methods for antioxidant activity assay of four Romanian herbs

The present study investigates the antioxidant activities of some Romanian plants, using different spectrophotometric methods (FRAP I, FRAP II, and CUPRAC). The plants investigated are hawthorn (Crataegus oxyacantha), bilberry (Vaccinium myrtillus L.), rosehip (Rosa canina), and chokeberries (Aronia melanocarpa). Hawthorn is used to treat a wide variety of inflammatory conditions, but the primary use is generally restricted for treating hypertension, ischemic heart disease, congestive heart failure, and arrhythmia. Investigations have proved the safe and reliable use of plant and plant extracts for treatment of cardiovascular disorders.     

A spectrophotometric assay for quantification of artemisinin

A sensitive and rapid spectrophotometric method for determination of artemisinin concentration is described. The method is based on the measurement of a reaction product of the drug in strong alkali solution. The interaction produces a homogenous electronic transition band from 250 to 330 nm with maximum transition at around 291 nm. The absorption curve shows Gaussian distribution with identical half bandwidth, thus providing information for formation of a possible mono-type reaction product. The 291 nm absorption intensity increases with increasing concentration of artemisinin and obeys Beer's law in the range of 0.44-172 nmol (ml⁻¹). The optimum reaction conditions and other analytical parameters were evaluated including its recovery from human plasma and erythrocyte samples.     

Simple and rapid enzymatic assay of ornithine decarboxylase activity

Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.     

A new spectrophotometric method for assay of chloramphenicol

Summary Ninhydrin in the presence of SnCl₂ in 0.2M citrate buffer (pH 5) is proposed as a new reagent for the Spectrophotometric assay of chloramphenicol (CP), CP esters and CP in pharmaceutical formulations such as tablets, capsules, eye drops, eye ointments, ear drops and injections. The method is based on hydrolysis of chloramphenicol with 4M NaOH and subsequent treatment with ninhydrin reagent. The resulting violet colour exhibits maximum absorption at 570 nm. The Beer's law limits, molar absorptivity, effect of time and reagent concentration, and interference studies are reported.

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Eine neue spektrophotometrische Methode zur Bestimmung von Chloramphenicol

Zusammenfassung Für die spektrophotometrische Bestimmung von Chloramphenicol (CP) und CP-Estern in pharmazeutischen Präparaten (Tabletten, Pillen, Augentropfen, Augensalben, Ohrentropfen und Injektionspräparaten) wurde als neues Reagens Ninhydrin mit SnCl₂ in 0,2M Citratpuffer (pH 5) vorgeschlagen. Das Verfahren beruht auf der Hydrolyse von CP mit 4M NaOH und nachfolgender Behandlung mit dem Ninhydrinreagens. Die sich dabei ergebende Violettfärbung hat ihr Absorptionsmaximum bei 570 nm. Die Gültigkeitsgrenzen des Beerschen Gesetzes, die molare Absorbanz, der Einfluß der Zeit und der Reagenskonzentration sowie die Störfaktoren wurden angegeben.

     

A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases

A fast and sensitive method for screening transaminase activity and enantioselectivity, using D- and L-amino acid oxidases, allows new amine substrates to be rapidly identified.     

A lab-on-a-chip for spectrophotometric analysis of biological fluids

This paper reports a lab-on-a-chip for application in clinical analysis, especially in the spectrophotometric analysis of biological fluids. It is composed of three parts: (1) a microfluidic system die containing the microchannels fabricated using SU-8 techniques; (2) an optical filtering system based on highly selective Fabry-Perot optical resonators using a stack of CMOS process compatible thin-film layers; (3) a detection and readout system fabricated in a CMOS microelectronic process. The system enables low-cost and selective measurement of the concentration of several biomolecules in biological fluids. Operation is based on optical absorption in a well-defined part of the visible spectrum, defined by the reaction of a specific reagent with a specific biomolecule. Signals proportional to the intensity of the light transmitted through the biological fluid are available at the output in the form of bit streams, which allows simple computer interfacing. Moreover, the optical filtering system enables the measurement using white light illumination, thus avoiding the use of a wavelength dependent light source. This characteristic makes the lab-on-a-chip portable and ensures that the analysis can be performed at any location with instantaneous results, without the use of complex and expensive analysis systems. The quantitative measurement of uric acid and total protein in urine is demonstrated.     

Intralaboratory validation of cell-free translation assay for detecting ricin toxin biological activity

A cell-free translation (CFT) assay for determining ricin biological activity was validated. The statistical data from the validation study showed a high level of precision within and between runs of the assay. The assay was specific for determining ricin biological activity in food-based matrixes and discriminated ricin from other ribosome-inactivating proteins. The mean bias (relative error) between measured ricin concentrations of 3 validation samples and their nominal concentrations was 1.1, 6.6, and 20.3%, while the coefficient of variation (CV) was 14.1, 7.7, and 13.5%, respectively, demonstrating good precision, accuracy, and linearity. The CVs of ricin concentrations in 2 ricin-containing samples calculated from a dilution series were <5 and <12%, respectively, demonstrating very good parallelism. The analyte stability of ricin-containing samples stored for 1 month either at 4 or -20 degrees C, the stability of ricin stock solutions, and the results of assays executed by different analysts and using different luminometers were evaluated. The statistical validation data confirmed that the 4-parameter logistic equation, y = (a - d)/[1 + (x/c)b] + d, provided an accurate representation of a sigmoidal relationship between the measured response and the observed ricin concentration for the CFT assay.     

A rapid spectrophotometric method for iodimetric α-amylase assay

A kinetic, two-point method for the assay of α-arnylase in serum, involving spectrohotometric measurement of a starch-iodine complex, is described. This approach avoids interferences by serum proteins and other substances that react with iodine. The method requires less than 4 min per assay, only 10 μl of sample is used, and precision and accuracy are comparable to those of established procedures.     

A spectrophotometric assay for solid phase primary amino groups

A rapid, sensitive, and convenient spectrophotometric assay was developed for the measurement of amino groups on solid supports. This method is based on the reaction of amino groups of solids with an excess ofo-phthaldialdehyde (OPA) and subsequent quantitative determination of unreacted OPA by reaction with glycine. Four solids possessing variable quantities of amino groups were examined. Results indicate that about 70% of the total surface amino concentration (determined by the microKjeldahl method) are available for ligand attachment. Unlike the spectrophotometric 2,4,6-trinitrobenzenesulfonic acid method, the OPA spectrophotometric assay is more rapid, sensitive, and convenient, and unlike the spectrofluorimetric OPA, it does not require sophisticated instrumentation.     

A spectrophotometric assay for biotinbinding sites of immobilized avidin

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB₁. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 μmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.