Dual wavelength/stopped flow spectrophotometry: computer acquisition and analysis.

The adaptation of a commercially available dual wavelength/stopped flow spectrophotometer for use with turbid samples is described. A minicomputer is used to collect and analyze the data, thereby facilitating these experiments. The stopped flow/computer combination has a dead time in the single wavelength mode of 3.5 msec. In the dual wavelength mode, accurate determinations can be made of the time course of reactions that have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 50 msec or longer. The application of this stopped flow spectrophotometer to the measurement of cytochrome P-450 reductase activity in rat liver microsomes is described.     

A high performance micro-dual-wavelength-spectrophotometer (MDWS)

The dual wavelength spectrophotometer (DWS) has proven to be the most sensitive device to monitor minute optical absorbance changes, which are inaccessible to conventional single or double beam spectrophotometers. The typical set ups, e.g. extensively used for Ca2+ or phytochrome measurements, are huge, expensive and cumbrous. Therefore, a novel high performance micro-dual-wavelength spectrophotometer (MDWS) was developed. It is miniaturized and no moving parts such as vibrational mirrors or rotating filter wheels involved. Its specifications are superior compared to the conventional set up being capable of detecting minute optical changes (reflection, absorbance, transmittance) at particular wavelengths.     

Computer-assisted indirect coulometric titrations of biological redox components

The optically coupled, indirect coulometric titration (ICT) method has been applied to assist in the characterization of several redox biocomponents such as heme proteins. This method has provided rapid and repetitive evaluation of redox stoichiometry and energeties with a high degree of precision. The usual manual ICT procedure requires operator control of the repetitive incremental additions of electrochemical charge, the on/off of the magnetic stirrer between such additions, and the recording of optical spectra after each addition. The sequence and timing of events in the above procedure can be fixed which then lends itself ideally to computer control. In this paper, a computer-controlled ICT intsrumentation is described. The hardware and software developed for the computerization permit versatility of displaying ICT data in various formats, i.e., conventional absorbance or derivative spectra and dual wavelength or differences between dual-wavelength spectra. Also, absorbance at a given wavelength could be obtained from the spectra as a function of the incremental addition of charge. The three-dimensional plots of spectral absorbance as a function of wavelength and incremental charge could also easily be obtained. To demonstrate the capability and versatility of the computer-controlled instrumentation, the ICT of the redox components, cytochrome c and cytochrome c oxidase, and of intact mitochondria is presented.     

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

A polychromatic flash photolysis apparatus (PFPA)

A wide variety of biologically relevant chemical intermediates have been identified and characterised by their spectral properties. When rapid kinetics, i.e. rapid changes in these spectral properties are studied, the equipment designed for these studies (flash photolysis-, T-jump apparatus) usually allows only the registration of intensity changes of the monitoring light beam at one particular wavelength. Quite frequently, however, particularly in biological systems, the reactions of interest are too complex to be fully understood using single wavelength techniques. We have therefore designed and built a flash photolysis apparatus which permits the simultaneous recording of absorbance changes at 32 wavelengths, freely selectable between 300 and 1000 nm, as well as changes in fluorescence, luminescence, birefringence and light scattering. The apparatus, which we have called Polychromatic Flash Photolysis Apparatus (PFPA), acquires up to 8000 difference spectra per second with an amplitude resolution of better than 0.0001 absorbance unit. Data acquisition and activation of an actinic xenon flash unit occurs under computer control. The same computer is responsible for data storage, handling and graphic display. This communication describes the PFPA, its performance, and, as a demonstration of its potential usefulness, its application to the measurement of the light driven photocycle of bacterial rhodopsin, the proton pumping protein of Halobacterium halobium.     

A scanning dual wavelength spectrophotometer: Application to the study of photosynthetic electron transport

A dual wavelength scanning spectrophotometric method for the analysis of small absorbance changes in turbid suspensions is presented, which improves upon the point by point method of obtaining difference spectra in terms both of accuracy and rapidity. Examples are shown of the application of this instrument, in conjunction with a computer curve-resolving routine, to problems in photosynthetic electron transport.     

A double-beam rapid-scanning stopped-flow spectrophotometer.

A double-beam rapid-wavelength-scanning stopped-flow spectrophotometer system based on the Norcon model 501 spectrometer was construced, which enables u.v.-or visible absorbance spectra to be recorded at the rate of 800/s after the rapid mixing (within 3ms) of two reactant solutions. Each spectrum spans about 200nm in 1ms. It is possible to record difference spectra during reactions with half-lives less than 10ms involving absorbance changes of less than 0.1 absorbance unit. Analogue circuitry is used to produce spectra of absorbance against wavelength. Up to 32 such spectra can be recorded at pre-selected times during a reaction and stored in an 8Kx8-bit-word hard-wired data-capture system to be subsequently displaned individually or simultaneously. Time-courses at different wavelengths can also be displayed. By averaging up to 216 spectra it is possible to record spectra under conditions of low signal-to-noise ratios...     

The use of an internal standard for semiquantitative analysis of low temperature (77°K) fluorescence of photosynthetic cells

A simple method is described which permits quantitative estimation of chlorophyll fluorescence emission intensity relative to the chlorophyll concentration in sample material at 77°K. A fluorescent standard excited by light of a similar wavelength as that of the sample material but emitting at a slightly shorter wavelength is mixed with the sample in known proportions. The fluorescence yield of the various peaks of the sample are normalized to the fluorescence yield of the internal standard. The spectra are recorded using an inexpensive attachment consisting of a Dewar holder, mounted instead of the light source of any double beam or split beam spectrophotometer. A glass rod is immersed in the sample standard mixture and then placed in the liquid nitrogen containing Dewar. Exciting light is provided by a light pipe mounted at 90° to the sample holder, connected with a tungsten-hallogen lamp provided with an appropriate filter. This method has been found to be particularly useful for the quantitation of chlorophyll fluorescence emission at 77°K of photosynthetic cells or chlorophyll containing membrane fractions. For this purpose, phycocyanin was found to be a suitable internal standard.     

Arsenazo III-Ca2+. Effect of pH, ionic strength, and arsenazo III concentration on equilibrium binding evaluated with Ca2+ ion-sensitive electrodes and absorbance measurements.

Equilibrium binding properties of the metallochromic indicator Arsenazo III (AIII) were characterized by Ca2+ and acid/base titration. Free calcium was measured directly with Ca2+ ion-sensitive electrodes. Absorbance changes were measured by both a conventional scanning spectrophotometer and a dual wavelength spectrophotometer. Acid/base titration of AIII in conjunction with Ca2+ ion-sensitive electrode measurement and absorbance changes indicate that pH can change AIII absorbance through a change of the K(D) for Ca-AIII formation, and, in addition, that there is a pH-specific component that is not dependent on Ca-AIII formation. THe dissociation constant (K(D)) of AIII varied not only with pH, but with ionic strength and AIII concentration. Studies conducted to examine AIII-Ca stoichiometry resulted in different initial conclusions, depending on the method of analysis. Log delta A-log AIII relations were in accord with previously published results, which indicate that more than one AII binds to one Ca2+ ion. But Job plots. Scatchard analysis, and Hill plots all indicated 1:1 binding. THe method of absorbance measurement, i.e., scanning or dual wavelength did not influence the results. these findings were reconciled on the basis of changes in K(D) with AIII concentration and ionic strength. In 200 mM KCl, K(D) of AIII-Ca varies by a factor of 7 between 10(-4.3) and 10(-3) M AIII. Thus, a disproportionately large amount of Ca-AIII is formed as AIII concentration is increased, which results in slopes greater than unity for log delta A-log AIII relations.     

New multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam for photosynthesis research

A multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam and differential optics has been constructed for measurements of light-induced absorbance and fluorescence yield changes in isolated chlorophyll-proteins, thylakoids and intact cells including algae and photosynthetic bacteria. The measuring beam, provided by a short (2 μs) pulse from a xenon flash lamp, is divided into a sample and reference channel by a broad band beam splitter. The spectrum in each channel is analyzed separately by a photodiode array. The use of flash measuring beam and differential detection yields high signal-to-noise ratio (noise level of 2 × 10⁻⁴ in absorbance units per single flash) with negligible actinic effect. The instrument covers a spectral range between 300 and 1050 nm with a spectral resolution of 2.1, 6.4 or 12.8 nm dependent on the type of grating used. The optical design of the instrument enables measuring of the difference spectra during an actinic irradiation of samples with continuous light and/or saturation flashes. The time resolution of the spectrophotometer is limited by the length of Xe flash lamp pulses to 2 μs.     

Electrophoresis with continuous scanning densitometry: separation of cells in a density gradient.

An instrument and procedure for electrophoresis with continuous optical scanping densitometry, automated data processing, and related methodology are described for the continuous analysis of electrophoresis and unity gravity sedimentation of macromolecules or cells in a static density gradient system. The instrument consists of a dual-beam spectrophotometer, a scanning stage and scanner control unit, an electrophoresis cell cassette, a filling/purging/cooling module, an analog-to-digital converter, and a digital data-logger. The distribution of cells is monitored repetitively during migration by absorbance measurements at any wavelength in the 200–800-nm range. A computer program provides the statistical analysis of each peak (baseline correction, smoothing, area, mean, standard deviation, skewness, and kurtosis) which can be further utilized for computing additional parameters, such as resolution and heterogeneity. A mixture of human and rabbit erythrocytes were used as a model system to evaluate the performance of the instrument and demonstrate some of its capabilities.     

An inexpensive computerized enzyme kinetics system based on a Gilford spectrophotometer and an Apple IIe microcomputer

A microcomputer-controlled data acquisition system for spectrophotometric enzyme kinetics measurements has been assembled. The system uses an Apple IIe computer which is interfaced to the binary coded decimal output of a Gilford spectrophotometer. No analog-to-digital converter had to be purchased. A BASIC program which collects timed absorbance readings every 500 ms, plots the data in real time, performs a linear regression of the data to measure the reaction rate, and calculates the enzyme activity concentration is given in full. Details describing the interfacing of the computer to the spectrophotometer are presented which will permit other laboratories to readily assemble their own systems using this hardware. Kinetic data acquired by the system are highly reproducible and agree well with data processed much more slowly by manual techniques from strip chart recordings.     

A protein and peptide monitor with a hollow-cathode light source.

This communication describes an inexpensive system that will monitor protein and peptide concentration in chromatogram eluates by light absorbance at an adjustable wavelength.Proteins in chromatogram eluate streams are commonly metered for concentration by absorbance measurement at 280 nm. Besides a range of commercially manufactured monitors, there is the apparatus described by Bennett et al. (1) which uses a selectively modulated magnesium lamp. Measurements in the region of 280 nm are of no value, however, when the material does not contain aromatic amino acids. Monitoring then becomes necessary at 230 nm in the region of absorption due to the peptide bond. The common resort in such a case is the standard ultraviolet-visible spectrophotometer, which has the disadvantage of being both unnecessarily elaborate and expensive for the purpose required. The deuterium lamps in these instruments require frequent replacement because of the extended periods of operation, adding to the cost factor.We have investigated the use of a hollow-cathode lamp of the type manufactured for use in an atomic absorption spectrophotometer. These lamps have high stability and a long working life, due to the considerably lower level of power dissipation compared with deuterium lamps. Their emission spectra are discontinuous, but the lamp for the element iron provides adequately strong lines at both 229.5 and 279.2 nm, suitable for a protein monitor.     

LED array spectrophotometer for measurement of time resolved difference spectra in the 530–600 nm wavelength region

A new type of computer controlled spectrophotometer is described which is based on an array of independent, monochromatic pulsed light sources consisting of light emitting diodes (LED) equipped with narrow band interference filters. The LEDs are sequentially pulsed at a high repetition rate. The absorbance information at specific wavelengths is sampled in the s-time range, using a computer-controlled, highly selective technique of synchronous amplification. A first prototype of this LED Array Spectrophotometer allows simultaneous recording of kinetic changes at 16 different wavelengths in the range from 530 to 600 nm, with a time resolution of 1 ms/point. Special features of the new type of spectrophotometer are: Weak integrated measuring light intensity, high signal/noise ratio even with scattering samples like intact leaves, active baseline adjustment by LED current regulation, computer control of system operation and data analysis. To deconvolute the complex absorbance changes in the cytochrome -band region, standard spectra of the major components are stored in computer memory and used for curve fitting of difference spectra and kinetic changes. As an example of application, the light-induced absorbance changes in a heat-pretreated spinach leaf are analysed. The system effectively separates specific absorbance changes of C550, cyt f, cyt b ₅₅₉ and cyt b ₅₆₃ from a large background of non-specific changes.Abbreviations DCMU 3-(3, 4-dichlorophenyl-)1, 1-dimethylurea - DAD diaminodurol - DNP-INT 2, 4-dinitrophenylether of 2-iodo-4-nitrothymol - ANT-2p 2-(3-chloro-4-trifluoromethyl)-anilino-3, 5-dinitrothiophene - RAM random access memory - LED light emitting diode - HBW half bandwidth     

A semiautomated system for measurement of 96 simultaneous spectrophotometric enzyme assays

A semiautomated system for spectrophotometric measurement of enzyme activity is described. In comparison to a 1-ml reaction volume monitored continuously by a conventional spectrophotometer, this system requires 1/10 to 1/100 the volume of sample, and 1/8 to 1/4 the time for measurement and computation of 96 enzyme assays. The system hardware consists of a 96-well platereader interfaced to a personal computer. Absorbances of 96 reactions are measured at timed intervals. These data are transmitted electronically from the platereader to the computer through the modem port using a modem program. The reaction rates are computed from the timed absorbance readings using a spreadsheet program. Three enzyme assays are presented, but the method has been used for several other assays and is applicable to many spectrophotometric rate assays. Many laboratories currently possess one or both of the two major components of the relatively inexpensive system described.     

A computer-controlled laser Raman spectrophotometer with interactive-graphics data analysis.

The computerization of a laser Raman spectrophotometer is described which permits automated operation of the instrument for signal averaging. The use of an interactive-graphics computer terminal for the rapid reduction of digitized data is discussed and illustrated by the acquisition and analysis of the Raman spectrum from the enzyme, protocatechuate 3,4-dioxygenase.     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

Quantacorrected Spectrometer for Recording Different Types of Photobiological, Photochemical, and Spectrophotometric Measurements

An instrument is described which functions as a spectrophotometer, In addition it measures action spectra of photobiological processes, excitation and emission spectra of fluorescence, and gives data for calculation of action spectra of partial reactions of photosynthesis and photocontrolled enzymatic reactions. The instrument has a Bausch and Lomb 250 mm grating monochromator. Equal numbers of quanta at different wavelengths are adjusted electronically with control of the slit through a quantacorrected photocell and a servo motor which is programmed by a cam connected to the wavelength drive.     

On-line determination of pigment composition and biomass in cultures of microalgae

Summary Rhodomonas sp. was grown in a photo-bioreactor equipped with a measuring cell in a spectrophotometer as part of an external flow loop. The apparent absorbance from 400 to 800 nm of the cell suspension was recorded at predetermined intervals and stored in a computer. From the spectra, the biomass and the concentrations of the two pigments chlorophyll a and phycoerythrin were determined in nitrogen-limited batch cultures.     

Manometric and spectrophotometric procedures for measurement of procaine hydrolysis by mouse liver in vitro

A decrease in absorbance at 313 nm was used to determine procaine hydrolysis by mouse liver. Results suggested that procaine hydrolysis was not linearly dependent upon the amount of tissue added to the incubate. This resulted from the fact that p-aminobenzoic acid, one of procaine's hydrolysis products, also absorbed at 313 nm. A dual wavelength analysis procedure is described which permitted accurate measurement of procaine hydrolysis by mouse liver without physical separation of procaine from its hydrolysis products. Measurements by the dual wavelength and manometric methods of inhibition of procaine hydrolysis in livers from mice treated with triorthotolyl phosphate indicated that both methods yielded quantitatively comparable results. The dual wavelength procedure is recommended for future studies of procaine hydrolysis by mammalian tissues.