Spectrophotometric determination of H2O2-generating oxidases using oxyhemoglobin as oxygen donor and indicator

1. Spectrophotometric determination of oxygen uptake using oxyhemoglobin as oxygen donor and indicator was used for assay of H2O2-generating oxidases like monoamine oxidase and glucose oxidase. 2. In order to decompose H2O2 formed during the oxygen uptake, catalase and methanol (or ethanol) was added to the respiratory system. At pH values higher than 7.5 the oxydation of deoxygenated hemoglobin to methemoglobin was less than 3%. 2. Oxidases with low Km for oxygen can be assayed using the spectrophotometric method if suitable correction factors are introduced into the calculation of oxygen uptake. The correction factor represents the ratio of the rate of formation (or disappearance) of one of the reactants and the rate of oxyhemoglobin deoxygenation, measured under identical experimental conditions.     

A new spectrophotometric cuvette holder for low temperature studies; Its application to the study of carbonmonoxyhemoglobin oxidation rate

A new spectrophotometric cuvette holder to be used for subzero temperature is described. The device is easily adaptable to a commercial spectrophotometer and it was checked down to --40 degrees C. Satisfactory mixing of the reactants contained in the cuvette at low temperatures is attained using a special stirrer and suitable solution volumes. The rate of carbonmonoxyhemoglobin oxidation by K3Fe(CN)6 at different subzero temperatures has been studied using this apparatus; the results are in agreement with the extrapolated data at room temperature.     

Oxygen consumption in Plasmodium berghei-infected murine red cells: a direct spectrophotometric assay in intact erythrocytes

A spectrophotometric assay has been devised to measure oxygen consumption non-invasively in intact murine red cells parasitized by Plasmodium berghei. The method uses oxyhemoglobin in the erythrocytes both as a source of oxygen and as an indicator of oxygen consumption. Spectra of intact cells show broad peaks and sloping baselines due to light-scattering. In order to ascertain the number of varying components in the 370-450 nm range, the resolution of the spectra was enhanced using Fourier transforms of the frequency domain spectra. Calculation of oxygen consumption was carried out for two-component systems (oxyhemoglobin, deoxyhemoglobin) using absorbances at 415 and 431 nm. Samples prepared from highly parasitized mice (greater than 80% parasitemia, 5% hematocrit) showed oxygen consumption rates of (4-8) X 10(-8) microliter/cell per h. This rate was not attributable to the presence of white cells or reticulocytes. The rate of oxygen consumption in the erythrocytes is shown to be modulated by various agents: the respiratory inhibitors NaN3 and KCN (1 mM) reduced oxygen consumption 2-3-fold; salicylhydroxamic acid (2.5 mM) caused a 20% reduction in rate and 10 mM NaN3, completely blocked deoxygenation. Antimalarial drugs and metal-chelating agents were also tested. Chloroquine, EDTA and desferal (desferoxamine mesylate) did not decrease the deoxygenation rate of hemoglobin in parasitized cells. Quinacrine, quinine and primaquine reduced the rate of formation of deoxyhemoglobin but also produced substantial quantities of methemoglobin. The lipophilic chelator, 5-hydroxyquinoline, decreased the rate of deoxygenation one-third. The spectrophotometric assay provides a convenient means to monitor oxygen consumption in parasitized red cells, to test the effects of various agents thereon, and potentially to explore possible mechanisms for oxygen utilization.     

A new, rapid, and sensitive assay for adenosine 5'-phosphosulphate (APS) kinase.

A new method for the determination of adenosine 5′-phosphosulphate (APS) kinase activity using a spectrophotometric assay is described. This procedure involves the spectrophotometric determination of sulphate- or APS-dependent production of ADP in the presence of pyruvate kinase and lactate dehydrogenase. Methods are described that overcome interference from contaminating enzymes and compounds. This method also provides a means for a critical examination of the substrate specificity of the sulphate-activating enzymes.     

Generation of active oxygen species during coupled autoxidation of oxyhemoglobin and δ-aminolevulinic acid

The conversion of oxyhemoglobin to mathemoglobin has been shown via spectrophotometric, circular dichroism and polarographic studies to be accelerated by δ-aminolevulinic acid, a major heme-precursor accumulated in a number of heme-linked pathologies. Concomitantly, δ-aminolevulinic acid undergoes aerobic oxidation. The intermediacy of oxygen radicals in these processes was evidenced by the inhibitory effect of catalase, superoxide dismutase and mannitol. These results are relevant to the exacerbated production of active oxygen species in intermittent acute porphyria and saturnism carriers.     

A computerized spectrophotometric instrumental system to determine the "vertical velocity" of sperm cells: a novel concept.

BACKGROUND: The presently available cell motility-analyzers measure primarily the "horizontal" velocity and there is no instrument available for "vertical" velocity measurement. This development was based on the turbidimetric method of sperm motility analysis. METHODS: Sperm was layered at the bottom of the cuvette containing buffer solution and exposed to the spectrophotometric light path at different heights to track the vertically moving sperms. The vertical movement was materialized with the development of an electromechanical up-down movement devise for the cuvette accomplished with the help of a cuvette holder-stepper motor-computer assembly. The entire system was controlled by the necessary motion control, data acquisition, and data processing software developed for cuvette movement and data analysis. RESULTS: Using goat sperm as the model a unique computer-based spectrophotometric system has been developed for the first time to determine the average "vertical" velocity of motile cells. CONCLUSIONS: Undertaking upward movement against gravity is much tougher as compared with horizontal movement. Consequently average vertical velocity is expected to be a much better identifying parameter for assessing semen and other motile cell quality. The novel instrumental system developed by us has thus the potential for immense application in human infertility clinics, animal-breeding centres, centres for conservation of endangered species, and also for research work on vertical velocity of spermatozoa and other motile cells, such as bacteria, protozoa, etc.     

Spectrophotometric determination of antioxidant activity

Summary

The spectrophotometric technique for total antioxidant activity (TAA)^1,2 measures the relative abilities of antioxidants to scavenge the 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS^?+) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS^?+ as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.     

On the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. The wavelength dependence of observed oxygen equilibrium parameter values

Spectral changes of oxyhemoglobin induced by such anions as 2,3-diphosphoglycerate, inositol hexaphosphate, and Cl- may affect the validity of the spectrophotometric determination of oxygen saturation of hemoglobin. Therefore, the anion-induced difference spectra were extensively measured under a variety of conditions and accurate oxygen equilibrium curves were determined under representative conditions with detection at different wavelengths selected from peaks, troughs, and zero difference points of the difference spectra in the visible and Soret regions. Oxygen equilibrium parameters including the four Adair constants (i.e., equilibrium constants for four steps of oxygenation) estimated from the equilibrium curves did not show any dependence on wavelength within the limits of experimental error. These results indicate that anion-induced spectral changes do not invalidate the spectrophotometric determination of oxygen saturation and confirm the validity of the previous conclusions drawn in our series of studies on the effects of anions, pH and temperature on oxygen equilibrium parameters.     

Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer

A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes. The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system. The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals. For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min. Based on the above results further possibilities for the application of the system for a variety of purposes are discussed.     

Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin

A continuous titration of absorption differences is described. Equal volumes of the titration fluid are dispensed from two micrometer-driven Hamilton gas-tight syringes into two 1 × 1 × 4.5-cm cuvettes. These are placed in the reference and sample beam. Each cuvette stopper is equipped with a capillary inlet connected to a syringe and with a minimotor for continuous stirring. Details of the stirring device are given. The delivered volumes of titration fluid are sufficiently reproducible to allow titration of absorption differences as a function of chromophore concentration. The usefulness of this approach is tested with the binding of 4-methylumbelliferyl α-d-mannopyranoside and concanavalin A as a well-characterized system. It is applied to the binding of similarly labeled anti-t disaccharide with the lectin from peanuts. With both lectins, the change in molecular extinction coefficient of the ligand and the association constant, valid for the entire protein saturation range, were obtained. The results are identical to those from other methods, including equilibrium dialysis.     

Direct spectrophotometric analysis of hemoglobin in isoelectric focusing tube gels

Methods are described for the direct spectrophotometric analysis of human oxyhemoglobin, deoxyhemoglobin, and methemoglobin focused in polyacrylamide tube gels. Visible absorption spectra (350-650 nm) of electrofocused hemoglobin bands were recorded using a diode array rapid-scan spectrophotometer. Direct optical sampling of gels allowed the identification of focused hemoglobin valency hybrids which contain two oxidized monomers per tetramer.     

Modeling the enzymatic transformation of 3,5-dimethoxy,4-hydroxy cinnamic acid by polyphenoloxidase from the white-rot fungus Trametes versicolor

A mechanism for transforming sinapic acid by a polyphenoloxidase from Trametes versicolor was investigated using changes in sinapic acid and oxygen concentrations during the reaction. The experiments were performed in a closed system without supplemental oxygen. The effects of temperature and initial oxygen concentration on the reaction rates were examined. To compare the obtained results with those from spectrophotometric studies, some runs were performed using an open system with supplemental oxygen. Sinapic acid transformation can be described by the Theorell-Chance Bi-Bi or Ordered Bi-Bi mechanisms. This reacting system consisted also of additional enzymatic reactions between the products of sinapic acid transformation and oxygen. A mathematical model was developed using four ordinary differential equations that represent the Theorell-Chance Bi-Bi mechanism with three alternate substrates. Model parameters (i.e., rate constants) were determined using the data collected at three different temperatures. On the basis of the transition state theory, relationships between these constants and temperature were established. It is shown that, in the open system, the observed change in the enzyme activity at higher temperatures was caused by two opposing phenomena: an Arrhenius effect which increased the rate, and a solubility effect which reduced the rate due to a lower oxygen concentration. This finding allows us to recommend better conditions for spectrophotometric methods, the assay most commonly used to evaluate this and similar enzymes. (c) 1996 John Wiley & Sons, Inc.     

Fluorescence spot tests for DNA endonuclease, ligase, and topoisomerase activities

A rapid dilution cuvette has been designed for measurement of the rates of microtubule disassembly and the factors which stabilize tubules against dilution-induced depolymerization. The device minimizes microtubule shearing by obviating the passage of microtubules through small orifices, and optical measurements of the turbidity changes during disassembly are optimized by use of a long-path-length configuration. Details on the design and application of the rapid dilution cuvette to probe microtubule disassembly are described here. In principle, the method may be applied to study any indefinite polymerization process which is characterized by critical concentration phenomena.     

Enzymatic analysis of guanine nucleotides in tissues and cells

GTP and GDP concentrations can be determined by a simple and specific spectrophotometric assay that uses commercially available enzymes. The conversion of GTP to GDP catalyzed by nucleosidediphosphate kinase in the presence of ADP enables the subsequent use of guanylate kinase which is coupled with hexokinase and glucose-6-phosphate dehydrogenase as indicator enzymes. Guanylate kinase which is highly specific for GDP and 5′-GMP (Miech, R. P., and Parks, R. E., Jr. (1965) J. Biol. Chem.240, 351–357) is also used for the determination of 5′-GMP and of the sum of all acid-soluble guanine 5′-nucleotides. The latter are hydrolyzed by snake venom phosphodiesterase and assayed as 5′-GMP. The assays are highly reproducible with standard deviations of less than 2% when performed in the optimal range between 2 and 100 nmol of guanine nucleotide per cuvette. The sensitivity can be increased by use of dual wavelength measurements of fluorimetry or by following the generation of ATP with the luciferase-catalyzed luminescence. Contents of guanine nucleotides and of total nucleoside 5′-triphosphates were measured in liver, kidney, brain, and skeletal muscle of the rat. The effect of guanosine and of inhibitors of inosinate dehydrogenase (virazole and mycophenolate) on the level of GTP and GDP was examined in ascites hepatoma cells in suspension.     

A simple assembly for anaerobic spectrophotometric reaction kinetics studies.

Details are given for the construction and use of a simple spectrophotometer cuvetteclosure that makes possible the study of reaction rates and enzyme assays under anaerobic conditions. The preparation of solutions and assembly of the cuvette unit is carried out in a N2 atmosphere in a glove box, but the spectrophotometric studies are conducted in a spectrophotometer with no modification except for the cell compartment cover.     

Elemental analysis of solid microscopic samples in the flameless atomic absorption cuvette

Summary Solid microscopic samples are precisely located by means of a newly developed applicator under microscopic control in the center of a flameless graphite tube cuvette. The parts of the applicator that reach into the cuvette are made of quartz which can be cleaned by heating. The values for the K and Na content of samples such applied are highly reproducible. No contamination could be detected. Solid samples yield higher signals than liquid samples. A method of calibration is described which uses lyophilized pieces of egg albumin containing known amounts of K and Na as standards.     

Elemental analysis of solid microscopic samples in the flameless atomic absorption cuvette.

Solid microscopic samples are precisely located by means of a newly developed applicator under microscopic control in the center of a flameless graphite tube cuvette. The parts of the applicator that reach into the cuvette are made of quartz which can be cleaned by heating. The values for the K and Na content of samples such applied are highly reproducible. No contamination could be detected. Solid samples yield higher signals than liquid samples. A method of calibration is described which uses lyophilized pieces of egg albumin containing known amounts of K and Na as standards.     

Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.     

Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin

A small stopped-flow cuvette was built into a computer-controlled Cary 210 spectrophotometer. The enzymatic depletion of oxygen in solutions of hemoglobin and myoglobin was initiated by flowing the hemeproteins with the enzyme against a solution of the hemeproteins containing the appropriate substrate. The deoxygenation was homogeneous throughout the solution. Oxygen activity was calculated at each instant of time from the fractional saturation of Mb, determined from observations at the Hb/HbO2 isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO2 isosbestic wavelength. The spectrophotometer cycled between these two wavelengths during the deoxygenation. The deoxygenation of HbO2 was largely complete in 20-25 min, whereas the deoxygenation of MbO2 was allowed to proceed for about 1 h. This procedure eliminates equilibration of Hb solutions with a gas phase and replaces oxygen electrode readings with spectrophotometric sensing by Mb, providing essentially instantaneous determinations of oxygen activity and hence 250-500 or more independent data points per run. The Mb and Hb data vectors require several manipulations to correct for small relative displacements in time and for small non-isosbestic effects. Detailed consideration of the enzyme kinetics allowed oxygen activities to be determined in regions where Mb is a poor sensor. Studies of HbO2 deoxygenation as a function of wavelength show that the determination of the four Adair constants requires in addition the determination of three spectroscopic parameters. Values of the apparent Adair constants, determined without these spectroscopic parameters, depend strongly on the monitoring wavelength.