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PubMed1985

Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles.

A rapid reconstitution procedure has been developed to insert deoxycholate-purified bacteriorhodopsin (bR) into asolectin vesicles. The procedure relies on the ability of the hydrophobic resin Bio-Beads SM-2 to remove octyl glucoside from a mixture of deoxycholate-purified bR, asolectin, and the detergent. Light-dependent acidification of the vesicle interior is observed with the reconstituted preparations as judged by the fluorescence quenching of an entrapped pH indicator, pyranine. Inhibition of proton pumping by the addition of LaCl3 to the external medium indicates that approximately 90% of the bR is oriented such that it pumps protons into the vesicles. Phase-lifetime spectrophotometry was used to study the relaxation processes associated with the intermediate in the photocycle of the reconstituted bR which absorbs at 410 nm. Amplitude spectra indicate that these absorbance changes are associated with the M intermediate in the bR photocycle. Two relaxation processes are observed. One is characterized by a relaxation time of approximately 4 ms and is independent of pH over the range 4.4-9.4. The longer relaxation time varies from 4 to 200 ms in the same pH range. By digitization of transients, which are observable when the actinic source is modulated at a low frequency, information about the dependence of the slower process on the light intensity and carbonyl cyanide m-chlorophenylhydrazone was obtained. The results can be interpreted in terms of two different forms of the M intermediate that decay on parallel kinetic paths. To explain the pH dependence of the decay rate, the slower decaying form must have three coupled protonation states, each with a different decay rate.

Bacteriorhodopsins↗
PubMed1981

Kinetics of pH-dependent interconversion of tryptophanase spectral forms studied by scanning stopped-flow spectrophotometry.

Morino and Snell [Morino, Y., & Snell, E. E. (1967) J. Biol. Chem. 242, 5591-5601] previously showed that the relative amplitudes of the 337- and 420-nm absorption bands of tryptophanase depended on both pH and the nature of a required monovalent cation activator. An investigation of the kinetics of interconversion of the 337- and 420-nm forms following a rapid incremental increase (jump) or decrease (drop) in pH over the range of enzyme stability in 0.2 M KCl at 24 +/- 0.3 degrees C by scanning stopped-flow spectrophotometry showed three distinct time-dependent phases. They were (1) an abrupt phase which is complete in less than 6.5 ms, (2) a fast first-order interconversion of the 420- and 337-nm absorbances, and (3) a slow first-order process involving growth at 355 nm coupled to two decays centered at 325 and 430 nm in the incremental pH jumps and decay at 355 nm with concomitant growth at 430 and 290 nm in the incremental pH-drop experiments. The results of these experiments were analyzed in terms of a scheme involving enzyme forms E alpha, E beta, E beta H+, E gamma, E gamma H+, and E delta. The E alpha form predominates in the absence of activating monovalent cations and absorbs at 420 nm. Those in the beta manifold, E beta and E beta H+, also absorb at 420 nm while those in the gamma manifold, E gamma and E gamma H+, absorb at 337 nm. The form E delta absorbs at 335 nm. E beta H+ and E gamma H+ represent the protonated form of the enzyme in each manifold. Analysis of the abrupt phase showed no significant systematic changes in absorbance above 330 nm for either the pH-jump or pH-drop experiments. The fast second phase involves the first-order interconversion of the beta and gamma manifolds while the slow third phase describes the buildup or decay of the delta manifold. Presumably conformational changes control the rate of these interconversions. The pH dependence of the fast first-order beta to gamma conversion was described and evaluated in terms of five independent equilibrium and rate constants and three independent amplitude terms by simultaneously fitting the amplitude data and first-order rate constants to an equation describing the overall scheme with a nonlinear least-squares program KINFIT4 [Dye, J. L., & Nicely, V. A. (1971) J. Chem. Educ. 48, 443-448]. The pK for protonation of the beta form = 9.70 +/- 0.12, for protonation of the gamma form (337-nm absorber) = 6.77 +/- 0.10, and for the pH-dependent interconversion of the beta and gamma manifolds, pKa = 8.11 +/- 0.04. The computed equilibrium distribution among the four species of the beta and gamma manifolds showed that E beta H+ and E gamma predominate.

Drug Stability↗
PubMed1998

The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry.

The reaction of Pseudomonas fluorescens kynureninase with L-kynurenine and L-alanine has been examined using rapid-scanning stopped-flow spectrophotometry. Mixing kynureninase with 0.5 mM L-kynurenine results in formation of a quinonoid intermediate, with lambdamax = 494 nm, within the dead time (ca. 2 ms) of the stopped-flow mixer. This intermediate then decays rapidly, with k = 743 s-1, and this rate constant is reduced to 347 s-1 in [2H]H2O, suggesting that protonation of this intermediate by a solvent exchangeable proton takes place. Rapid quench experiments demonstrate that covalent changes in the cofactor occur, as pyridoxal 5'-phosphate is converted to pyridoxamine 5'-phosphate in about 30 mol % within 5 ms after mixing. Under single turnover conditions in the reaction of kynureninase with l-kynurenine, a transient shoulder absorbing at 335 nm is observed that may be a pyruvate ketimine intermediate. In contrast, the reaction of kynureninase with 0.5 mM l-kynurenine in the presence of 10 mM benzaldehyde results in the formation of a quinonoid intermediate (k = 67.4 s-1) with a very strong absorbance peak at 496 nm. The reaction of L-alanine with kynureninase exhibits the rapid formation (386 s-1 at 0.1 M) of an external aldimine intermediate absorbing at 420 nm, followed by slower formation of a quinonoid intermediate with a peak at 500 nm (k = 2.5 s-1). The 420 nm peak then decays slowly with concomitant formation of a peak at 320 nm corresponding to a pyruvate ketimine. These data demonstrate that quinonoid and ketimine intermediates are catalytically competent in the reaction mechanism of kynureninase, and provide additional support for our proposed mechanism for kynureninase from steady-state kinetic studies [Koushik, S. V., Sundararaju, B., and Phillips, R. S. Biochemistry 1998, 37, 1376-1382].

Bacterial Proteins↗
PubMed1999

Reactions of dimethylsulfoxide reductase from Rhodobacter capsulatus with dimethyl sulfide and with dimethyl sulfoxide: complexities revealed by conventional and stopped-flow spectrophotometry.

Improved assays for the molybdenum enzyme dimethylsulfoxide reductase (DMSOR) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substrates are described. Maximum activity was observed at pH 6.5 and below and at 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been used to investigate the reduction of the enzyme by DMS to a species previously characterized by its UV-visible spectrum [McAlpine, A. S., McEwan, A. G., and Bailey, S. (1998) J. Mol. Biol. 275, 613-623], and its subsequent reoxidation by DMSO. Both these two-electron reactions were faster than enzyme turnover under steady-state conditions, indicating that one-electron reactions with artificial dyes were rate-limiting. Second-order rate constants for the two-electron reduction and reoxidation reactions at pH 5.5 were (1.9 +/- 0.1) x 10(5) and (4.3 +/- 0.3) x 10(2) M-1 s-1, respectively, while at pH 8.0, the catalytic step was rate-limiting (62 s-1). Kinetically, for the two-electron reactions, the enzyme is more effective in DMS oxidation than in DMSO reduction. Reduction of DMSOR by DMS was incomplete below approximately 1 mM DMS but complete at higher concentrations, implying that the enzyme's redox potential is slightly higher than that of the DMS-DMSO couple. In contrast, reoxidation of the DMS-reduced state by DMSO was always incomplete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reoxidized by DMSO, was obtained. Brief reaction (less than approximately 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in the presence of excess DMS, a further slow reaction occurred aerobically, but not anaerobically, to yield a stable enzyme form having a lambdamax at 660 mn. This state (DMSORmod) retained full activity in steady-state assays with DMSO, but was inactive toward DMS. It could however be reconverted to the original resting state by reduction with methyl viologen radical and reoxidation with DMSO. We suggest that in this enzyme form two of the dithiolene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflicting published information for DMSOR from X-ray crystallography and to previous spectroscopic data for its reduced forms.

Benzyl Viologen↗
PubMed2000

Measurement of 2-thiobarbituric acid values in dark chicken meat through derivative spectrophotometry: influence of various parameters.

Several variables (kind of filter paper, amount of sample, antioxidant addition, stability of the spectrophotometric measurement, and handling and storage of samples) were found to influence 2-thiobarbituric acid (TBA) values in dark chicken meat when an acid aqueous extraction method with derivative spectrophotometry was used. Filter papers with larger pore diameter or increasing sample weight led to lower TBA values. After incubation of reaction mixtures at 70 degrees C and ice-cooling, tempering for 45 min at room temperature was necessary to stabilize the spectrophotometric measurement. Furthermore, addition of butylated hydroxytoluene (BHT) and ethylenediaminetetraacetic acid disodium salt (EDTA) in the early steps of the method prevented artifactual formation of TBA reactive substances during analysis. Vacuum packaging and storage of samples at -20 degrees C were useful to avoid sample oxidation. The method finally proposed has a coefficient of variation of 3.81 or 4.13% for raw or cooked samples, respectively.

Animals↗
PubMed2002

Simultaneous determination of parathion and p-nitrophenol in vegetable tissues by derivative spectrophotometry.

A first derivative spectrophotometric method has been developed for the determination of parathion and p-nitrophenol in vegetable tissues. Ethanol was used as solvent for extracting the compounds from the tissues and subsequently the samples were evaluated against a vegetable tissue blank, directly by derivative spectrophotometry. The simultaneous determination of these compounds can be carried out using the zero-crossing approach for parathion at 253.0 nm and for p-nitrophenol at 273.1 nm. In the samples each analyte was determined in the presence of one another in the ranges between 4.9 to 3883.5 microg g(-1) for parathion and 4.9 to 3285.3 microg g(-1) for p-nitrophenol. The detection limits (3a) were found to be 1.5 and 1.4 microg g(-1) for parathion and p-nitrophenol, respectively. The relative standard deviations were in all instances less than 1.8%. The proposed method was applied to the determination of the analytes in spiked leaves of corn. The results show a good recovery and they are in agreement with those obtained by polarography.

Calibration↗
PubMed1997

Simultaneous determination of ethinylestradiol and levonorgestrel in oral contraceptives by derivative spectrophotometry.

A method for determining ethinylestradiol (ETE) and levonorgestrel (LEV) in mixtures by first-derivative spectrophotometry is described. The procedure does not require any separation step. Measurements are made at the zero-crossing wavelengths and the calibration graphs are linear up to 26 and 33 micrograms ml-1 of ETE and LEV, respectively. The method was applied to the determination of both compounds in five different Spanish commercial low-dose oral contraceptives. Similar results were obtained by an HPLC method.

Chromatography, High Pressure Liquid↗
PubMed1997

Simultaneous determination of the colorants sunset yellow FCF and quinoline yellow by solid-phase spectrophotometry using partial least squares multivariate calibration.

A method for the simultaneous determination of the colorants Sunset Yellow FCF and Quinoline Yellow using solid-phase spectrophotometry is proposed. The colorants were isolated in Sephadex DEAE A-25 gel at pH 5.0, the gel-colorants system was packed in a 1 mm silica cell and spectra were recorded between 400 and 600 nm against a blank. Statistical results were obtained by partial least squares (PLS) multivariate calibration. The optimized matrix by using the PLS-2 method enables the determination of the colorants in artificial mixtures and commercial soft drinks.

Azo Compounds↗
PubMed1989

Simultaneous determination of tranylcypromine sulfate and trifluoperazine dihydrochloride in tablets by first- and fourth- derivative ultraviolet spectrophotometry.

A rapid and simple method for the simultaneous determination of trifluoperazine dihydrochloride and tranylcypromine sulphate by first- and fourth-derivative UV spectrophotometry is presented. The procedure consists of extraction in 0.2 m hydrochloric acid, filtration and measurement of the amplitudes of the first- and fourth-derivative spectra at 260 and 274 nm, respectively. A zero-crossing technique is used for the determination of trifluoperazine. Good linearity, accuracy, precision and selectivity were found, and the method is proposed for routine quality control purposes, even for the uniformity of contents test.

Spectrophotometry, Ultraviolet↗
PubMed1991

Simultaneous determination of toxic metabolites by linear combination derivative spectrophotometry.

A linear combination derivative spectrophotometric method is described. The method overcomes the problem of overlapping in derivative spectrophotometry and allows the maximum use of quantitative information. In addition, the method can be used to increase the selectivity, sensitivity and accuracy of the simultaneous analysis of multicomponent mixtures. The application of the method to the simultaneous determination of bongkrekic acid and toxoflavin, the toxic metabolites produced by Pseudomonas farinofermentans, is described.

Bongkrekic Acid↗
PubMed1992

Simultaneous determination of theophylline and guaiphenesin by third-derivative ultraviolet spectrophotometry and high-performance liquid chromatography.

Two methods are described for the simultaneous determination of theophylline and guaiphenesin in combined pharmaceutical dosage forms. The first method depends on third-derivative ultraviolet spectrophotometry, with the zero crossing technique of measurement. Third-derivative amplitudes at 222 and 278 nm were selected for the assay of guaiphenesin and theophylline, respectively. The second method is based on high-performance liquid chromatography on a reversed-phase column using a mobile phase of 0.01 mol dm-3 sodium dihydrogen phosphate-methanol-acetonitrile (8 + 2 + 1) (pH 5.5) with detection at 245 nm. Both methods showed good linearity, precision and reproducibility. The proposed methods were successfully applied to the determination of these drugs in laboratory-prepared mixtures and in capsules or elixir.

Chromatography, High Pressure Liquid↗
PubMed1993

Determination of mercury levels in human urine and blood by ultraviolet-visible spectrophotometry.

A method for the determination of mercury levels in biological samples, i.e., human urine and blood, has been developed using an incomplete cubane-type sulfur-bridged molybdenum aqua complex, Mo3S4(4+)(aq), with analysis by spectrophotometry. The primary advantage of the method is that when the 'complex' is dissolved in sulfuric acid it reacts directly with mercury, produced by the addition of a reducing agent to samples containing mercury(II), giving an intense coloration. Levels of mercury in the biological samples can be determined using the 'complex' dissolved in sulfuric acid. The detection limit for mercury(II) in urine and blood was 0.05 ppm at the wavelength of maximum absorption (lambda max = 556 nm).

Humans↗
PubMed2000

Derivative spectrophotometry in the analysis of mixtures of phenols and herbicides.

Derivative spectrophotometry (zero-crossing technique) was applied to the determination of selected phenols and herbicides in two-component mixtures. Methyl- and chlorophenols (3-methylphenol, 2,3- and 3,4-dimethylphenol, 2,5-, 2,6- and 3,4-dichlorophenol and 2,4,5-trichlorophenol) and triazine, uracil and urea herbicides (simazine, propazine, hexazinone, bromacil and metoxuron) were examined. The RSD values ranged between 0.05 and 4% and the recoveries obtained were between 97 and 110%. The developed derivative spectrophotometric method was also applied as a complementary technique for the separation of overlapping peaks of sample compounds obtained by HPLC with diode-array detection. Metoxuron and 3-methylphenol, metoxuron and 2,5-dichlorophenol and simazine and 2,6-dichlorophenol were determined simultaneously by this method at the level of 1 x 10(-3) g l-1.

Environmental Pollutants↗
PubMed2004

A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve system.

Monitoring extracellular acidification rates (ECARs) is important for the study of cellular activities, since it allows for the evaluation of factors that alter metabolic function, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. While the light addressable potentiometric sensor (Cytosensor Microphysiometer) has been the principal tool for ECARs measurement in the past, this work introduces a novel method that exploits an immobilized pH indicator on the surface of microcarrier beads (Sephadex) and is probed with a fiber optic coupled spectrophotometer. Likewise, live cells under investigation were also immobilized on microcarrier beads (Cytopore). These beads are metered, transported and monitored within a microfluidic system, termed as the Lab-on-Valve (LOV). Use of carrier beads in conjunction with Bead Injection Spectrophotometry and a Lab-on-Valve module (BIS-LOV), makes ECAR measurements reliable and automated. The feasibility of the BIS-LOV approach is demonstrated measuring ECARs of the mouse hepatocyte cell line, TABX.2S, grown on Cytopore beads packed within the central channel of the LOV system. These immobilized cells were perfused in a phosphate buffer carrier solution (capacity: 1 mmol L(-1), pH 7.4). Protons extruded from 10(5) to 10(6) cells were accumulated during a stopped flow period of 220 s followed by a pH measurement, detected by changes in absorbance of the pH indicator bonded to the microcarrier beads. Addition of metabolic inhibitors (sodium azide, oxamic acid) to the carrier buffer solution can induced an increase or decrease of the basal proton extrusion rate in a very reproducible manner. Comparison of the BIS-LOV technique to the Cytosensor microphysiometer and literature confirms the validity of this novel approach, highlighting its advantages and suggesting future improvements that will make the BIS-LOV a practical tool for routine ECARs measurement.

Animals↗
PubMed1984

The reaction of fully reduced cytochrome c oxidase with oxygen studied by flow-flash spectrophotometry at room temperature. Evidence for new pathways of electron transfer.

Absorption changes during the O2 reaction of reduced bovine cytochrome c oxidase were investigated by the rapid-reaction technique of flow-flash spectrophotometry in the Soret, visible and near-i.r. spectral regions. New features in the time courses of absorption change were observed relative to the earlier findings reported by Greenwood & Gibson [(1967) J. Biol. Chem. 242, 1782-1787]. These new features arise in the Soret and near-i.r. regions and allow the reaction to be described at all wavelengths as a composite of three exponential processes. There is a rapid O2-sensitive phase detectable in the Soret and visible region. The second phase has a rate that is somewhat less dependent on O2 concentration than is the fastest phase rate and is detectable in all three spectral regions. The rate of the third phase is almost independent of the O2 concentration and is also detectable in all spectral regions. Analysis of the three phases gives their rates and absorption amplitudes. The fast phase reaches a rate of 2.5 X 10(4) s-1 at the highest O2 concentration available at 20 degrees C, whereas the phase of intermediate rate is limited at a value of 7 X 10(3) s-1 and the slow phase rate is limited at 700 s-1. The ratios of the kinetic difference spectra for the fast phase and the slow phase do not correspond to the spectra of the individual haem centres. A branched mechanism is advanced that is able to reconcile the kinetic and static difference spectra. This mechanism suggests that some of the cytochrome a is oxidized along with cytochrome a3 in the initial O2-sensitive phase. In addition, the model requires that CuA is oxidized heterogeneously. This fits with the complex time course of oxidation observed at 830 nm while retaining CuA as virtually the sole contributor to absorbance at this wavelength.

Electron Transport↗
PubMed1998

Near-infrared spectrophotometry determined brain oxygenation during fainting.

During orthostatic hypotension we evaluated whether presyncopal symptoms relate to a reduced brain oxygenation. Nine subjects performed 50 degrees head-up tilt for 1 h and eight subjects were followed during 2 h of supine rest and during 1 h of 10 degrees head-down tilt. Cerebral perfusion was assessed by transcranial Doppler determined middle cerebral artery blood velocity (MCA vmean), while brain blood oxygenation was assessed by near-infrared spectrophotometry determined concentration changes for oxygenated (delta HbO2) and deoxygenated haemoglobin and brain cell oxygenation by the oxidized cytochrome c concentration (delta CytO2). During head-up tilt, six volunteers developed presyncopal symptoms and mean arterial pressure (88 (78-103) to 68 (57-79) mmHg; median and range), heart rate (96 (72-111) to 65 (50-107) beats min-1), MCA vmean (59 (51-82) to 41 (29-56) cm s-1), delta HbO2 (by -5.3 (-3.0 to -14.8) mumol l-1) and delta CytO2 were reduced (by -0.2 (-0.1 to -0.4) mumol l-1; P < 0.05). During tilt down the cardiovascular variables recovered immediately and delta HbO2 increased to 2.2 (-0.9-12.0) mmol L-1 above the resting value and also delta CytO2 recovered. In the nonsyncopal head-up tilted subjects as in the controls, blood pressure, heart rate, MCA vmean and brain oxygenation indices remained stable. The results suggest that during orthostasis, presyncopal symptoms relate not only to cerebral hypoperfusion but also to reduced brain oxygenation.

Adult↗
PubMed1998

Near-infrared spectrophotometry of the brain in cardiovascular surgery.

Neuropsychological and neurological deficits are still major causes of mortality and morbidity after cardiac operations and are thought to be caused by embolism and cerebral hypoxia. Near-infrared spectrophotometry (NIRS) is a promising method for non-invasive monitoring of cerebral oxygenation and hemodynamics. Different devices provide information on changes of oxygenated (HbO2) and deoxygenated hemoglobin (Hb), oxidized cytochrome aa3 (CytOx) or regional oxygen saturation (rSO2). NIRS has been applied to patients during adult and pediatric cardiovascular surgery with and without deep hypothermic circulatory arrest (DHCA). In most of the studies, significant changes in cerebral oxygenation were detected by NIRS. NIRS measurements were influenced by the cerebral oxygen metabolism and the operative management. However, clinical, experimental, and theoretical issues raise doubts as to the clinical relevance of the hemoglobin saturation (HbO2, Hb, rSO2 signals) during hypothermia and alkalosis, because the oxygen affinity of hemoglobin increases and a high saturation might simply reflect the inadequate oxygen transport into cells. In contrast, recent experiments have proved a high correlation between the CytOx signal and the MRS parameters nucleoside triphosphate and phosphocreatine. Histological damage was significantly related to the lowest CytOx value; in a clinical study it predicted impaired neuropsychological outcome. Therefore, the CytOx signal is of great interest for future studies. NIRS must prove its ability to diagnose cerebral hypoxia consistently during cardiac surgery in a large patient study before this method is brought into routine clinical practice. Absolute quantification and definitions of critical oxygenation margins will be helpful for this goal.

Adult↗