Micromechanical measurements on P-protein aggregates (forisomes) from Vicia faba plants.

Forisomes are chemomechanically active P-protein aggregates found in the phloem of legumes. They can convert chemical energy into mechanical work when induced by divalent metal ions or changes in pH, which control the folding state of individual forisome proteins. We investigated the changing geometric parameters of individual forisomes and the strength and dynamics of the forces generated during this process. Three different divalent ions were tested (Ca2+, Sr2+ and Ba2+) and were shown to induce similar changes to the normalized length and diameter. In the concentration range from 0.1 to 4 M, K+ and Cl(-) ions had no influence on the contraction behaviour of the forisomes induced by 10 mM Ca2+. In the absence of dissolved oxygen, these changes were independent of the radius of the metal ion, water uptake and the strength of binding between the selected metal ions and those protein molecules responsible for forisome conformational transformation. In the absence of any load, bound Ca2+, Sr2+ and Ba2+ ions showed apparent and averaged dissociation constants of 14, 62 and 1070 microM, respectively. Various forisomes generated bending on a quartz glass fibre with a diameter of 9 microm. The fibre bending was measured microscopically also by correlation between the digital patterns of a predefined window of observation before and after bending. Similar bending forces of approximately 90 nN were measured for a single forisome sequentially exposed to 10 mM Ca2+, Sr2+ and Ba2+. In the absence of dissolved oxygen, the same conditions resulted in averaged bending forces of (93+/-40) nN, (58+/-20) nN, and (91+/-20) nN after contacting different forisomes with 10 mM Ca2+, 10 mM Sr2+, and 10 mM Ba2+ respectively, demonstrating that the force generated was independent on ion concentrations above a certain threshold value.

In vitro investigation of the geometric contraction behavior of chemo-mechanical P-protein aggregates (forisomes).

We investigated the contracting behavior of forisomes from Vicia faba by carrying out precise measurements of their changing geometric parameters in vitro in the absence and in the presence of dissolved oxygen. Furthermore, we investigated the fine structure of forisomes by scanning electron microscopy. For the first time, single forisomes were titrated with Ca(2+), protons, and hydroxide ions recording the complete progression of their contractions. An apparent Ca(2+)-binding constant of (22+/-3) muM was calculated from two complete titration curves. The forisomes also contracted in the presence of Ba(2+) and Sr(2+) ions, but the amplitudes of contraction were smaller under the same measuring conditions. The time taken to change from the longitudinally expanded into the longitudinally contracted state was up to 2 s shorter in 10 mM Ca(2+) in comparison to 0.2mM Ca(2+). However, the contraction time was prolonged by decreasing the Ca(2+) concentration. In the absence of dissolved oxygen, the transition between the two final states of the forisomes was almost reversible and the amplitude of contraction remained almost constant during the first 25 contraction cycles. In the presence of dissolved oxygen the forisomes denaturated after a few cycles and lost their ability to contract, just after only a few cycles with 10 min in the contracted state. Denaturation of the forisomes occurred appreciably in the contracted state. We propose a cycle process to explain the thermodynamic basis of the Ca(2+)-induced contraction and its reversal by EDTA. Reducing the pH-value from 7.3 to 4.0 caused the forisomes to shorten by approximately 15%, while increasing the pH to 11.0 caused them to shorten by 28 to 30%. In both cases, the increases of the forisomes volume were greater than during the Ca(2+) induced contraction. The pH values of 4.7+/-0.3, and 10.2+/-0.2 marked the inflection points of the acid base titration of different forisomes.

Nanoporous aluminum oxide as a novel support material for enzyme biosensors.

To construct novel amperometric sensors for the detection of hydrogen peroxide and pyruvate, peroxidase and pyruvate oxidase were immobilized in self-supporting nanoporous alumina membranes those made by anodic oxidation. Pyruvate oxidase and other enzymes were enclosed in poly(carbamoylsulfonate) hydrogel and sucked into the nanoporous alumina structure before polymerization. The alumina membranes were investigated by scanning electron microscopy before and after the enzyme immobilization. In an amperometric flow detector cell, pyruvate and hydrogen peroxide were detected under flow injection analysis conditions in concentration ranges from 1 microM to 100 microM and 5 microM to 500 microM, respectively. The achieved operational stability showed that alumina membranes can be used to construct enzyme-modified electrodes.

Sensitive and selective flow injection analysis of hydrogen sulfite/sulfur dioxide by fluorescence detection with and without membrane separation by gas diffusion.

Highly sensitive and selective FIA flow injection analysis procedures for the determination of sulfite/hydrogen sulfite/sulfur dioxide were developed on the basis of an in situ-generated o-phthalaldehyde (OPA)/ammonium reagent and fluorescence detection. The highest sensitivity was achieved at an excitation wavelength of 330 nm, an emission wavelength of 390 nm, and at pH 6.5. Sulfite concentrations between 2.5 nM and 5 microM can be determined with relative standard deviations between 10.5 and 1.0% (n = 5, confidence level alpha = 0.05) by utilization of a reagent that contains 0.2 mM OPA and 0.4 M NH4Cl in 50 mM potassium phosphate buffer. A concentration of 0.1 mM sulfite can be selectively detected in the presence of thiosulfate, thioglycolate, tetrathionate, cysteine, and ascorbate. The fluorometric sulfite detection was combined with a membrane gas diffusion step to improve the selectivity with respect to nonvolatile fluorescing substances. The total sulfite content can be quantitatively separated as sulfur dioxide into an acceptor solution before its flow detection. Between 40 nM and 0.1 mM sulfite can be determined. After 1,000-fold dilution, the total sulfite content can be determined in white and red wines.

Calibrationless determination of creatinine and ammonia by coulometric flow titration.

A precise and sensitive working microflow titration procedure was developed to determine creatinine and ammonia in urine samples. This procedure is based on enzymatic conversion of creatinine, gas diffusional membrane separation of the released ammonia into an acid acceptor stream, and coulometric titration of ammonia with hypobromite. The hypobromite is formed after the electrogeneration of bromine in an electrolyte containing 1.0 M NaBr and 0.1 M sodium borate adjusted to pH 8.5. The electrolysis current follows a triangle-programmed current-time course. An amperometric flow detector records the resulting mirror symmetrical titration curves, which show two equivalence points. The analyte concentration is calculated from the time difference between the equivalence points. For quantitative conversion of creatinine and quantitative separation of present and released ammonia no calibration is necessary to get accurate results. Both ammonia/ammonium and creatinine were determined in the range between 2 microM and 2 mM with relative standard deviations between 3.0 and 1.0% (n = 5). High recoveries were obtained for the analysis of diluted urine samples for both creatinine and ammonia.

Rapid and selective determination of ammonium by fluorimetric flow injection analysis.

Selective and sensitive procedures for the determination of ammonium in river water and diluted urine were developed by using flow injection analysis equipment. The methods are based on the derivatization of ammonia with o-phthaldehyde (OPA) and thioglycolate under alkaline conditions. The formed isoindole derivative is detected fluorimetrically at an excitation wavelength of 415 nm and an emission wavelength of 485 nm. The derivatization only takes 15 to 20 s at room temperature to achieve the maximum sensitivity. The optimized OPA reagent shows a surprisingly high selectivity for ammonium in the presence of many primary amines. With respect to the analysis of turbid and fluorescent sample solutions the selectivity can be improved by separating the ammonia through a microporous membrane from the OPA reagent. Without this separation step ammonia can be detected in the range between 0.05 and 100 microM with excellent linearity. After the insertion of an optimized membrane separation cell ammonia can be determined in the linear range between 0.2 microM and 20 mM.

Precise and sensitive determination of nitrite by coulometric backtitration under flow conditions.

A novel method performing for coulometric backtitrations in flow channels was developed, which was applied for the precise and sensitive determination of low concentrations of nitrite. Under acid conditions nitrite is reacting stoichiometrically with hydrazine, the remained excess of which is backtitrated with electrogenerated bromine. The titration course is recorded either amperometrically or chemiluminometrically. Hydrazine can be determined precisely and accurately in the range between 0.1 microM and 1 mM without calibration. Nitrite is reduced by hydrazine according to the reaction 2 NO2- + 2 H+ + NH2-NH2 --> N2O + N2 + 3 H2O. Applying the amperometric and the chemiluminometric end-point location nitrite was determined accurately and precisely in the ranges 0.25-65 microM and 0.10-50 microM, respectively. The method was applied to the determination of nitrite in tap and river water.

Detection and characterization of phospholipase D by flow injection analysis.

A precisely working automated system for the investigation of phospholipases D (PLDs, EC 3.1.4.4) from plant and microbial sources with flow injection analysis (FIA) has been developed. The two versions of the FIA setup described are based on the oxidation of choline liberated from phosphatidylcholine by PLD action and catalyzed by choline oxidase and the chemiluminescence detection of hydrogen peroxide produced by this reaction. The correlation between this chemiluminescence signal and the PLD activity was linear in the range between 1 and 100 mU/ml PLD. The sampling frequency was 12 samples per hour. This method was used to compare three different PLDs from cabbage and microbial sources with respect to their pH optima, temperature stability, effectors, and v/[S]-characteristics.

On-line monitoring of an animal cell culture with multi-channel flow injection analysis.

A multi-channel flow injection analysis system was used for on-line monitoring of a continuous animal cell culture with high cell density. With this system, the glucose, lactate and glutamine concentration were determined using immobilized dehydrogenases, ammonium using an aqueous o-phthaldialdehyde solution. Glutamine concentration was determined on the basis of the difference between a glutamine and a glutamate measurement. To prevent disturbance of the measurement and pollution of the system, the analytes in the sample were separated from high molecular compounds by on-line dialysis. On-line gas dialysis was used to avoid interference of other amino groups with the ammonium determination. In addition, dialysis was used as a dilution step. The measurement time for all four components was 42 min. This time included a final washing period after the analysis cycle. The system was calibrated once a day. Two continuous cultivations of a hybridoma cell line immobilized in open-porous glass carriers were monitored, using a fluidized bed reactor as cultivation system. The concentration of glutamine, glucose and ammonium determined with the on-line FIA system were in good agreement with the off-line data determined once a day. Only the lactate data showed some deviation. The immobilized enzyme reactors could be used for up to 3000-5000 injections. During the first cultivation, lasting 200 h, the start up period of the reactor was monitored. The on-line measurements described much better the time-course of the concentrations than the off-line data. It was possible to estimate the growth rate of the cells in the micro-carriers by the on-line data. In the course of the second cultivation, which lasted almost 1000 h, the influence of the dissolved oxygen concentration on the cell metabolism was monitored. It was noted that a sudden change of the glutamine concentration in the feed caused a fast change of the consumption and production rate of the measured metabolites.

Multichannel flow-injection-analysis biosensor system for on-line monitoring of glucose, lactate, glutamine, glutamate and ammonia in animal cell culture.

The application of a chemiluminometric method for the on-line monitoring of a hybridoma cell culture is described. Enzyme sensors for glucose, lactate, glutamine, glutamate and ammonia, based on oxidase-catalysed reactions, were developed and connected to a flow-injection-analysis (f.i.a.) biosensor. H2O2 produced by the oxidase-catalysed enzyme reaction was detected by luminol chemiluminescence with a fibre-optic H2O2 biosensor. The system has been used to monitor animal cell cultures. A continuous hybridoma cell cultivation for the production of monoclonal antibodies is presented as an example. It was possible to monitor the bioprocess over a period of 15 days. A complete analysis of all five components could be performed within 42 min. The enzyme sensors were stable during the whole cultivation time without significant loss of activity. The computer-controlled biosensor f.i.a. works with good reliability. The precision for all five components ranged between 2.2 and 4.5%. It was possible to determine glutamine in one step using an anti-interference enzyme reactor. Endogenous glutamate was completely removed up to a level of 0.5 mM.

Determination of intracellular dehydrogenase activities using flow injection analysis.

Reproducible rapid methods for cell disintegration in miniaturized flow cells were developed. The disintegration of yeast cells by ultrasonics and ultraturrax in a miniaturized flow through chamber was compared with the mixer mill disintegration. The flow through chamber was combined with a sterile on-line sampling device to take samples from yeast cell suspensions and with an automated flow injection analysis (FIA) set-up, to determine the released enzyme activities. The FIA set-up was tested with the sequential determination of malate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase in mixtures. The automated FIA determination of the intracellular formate dehydrogenase of yeast cells (Hansenula polymorpha) was performed.

A PC-based titrator for flow gradient titrations.

This paper describes a PC (personal computer) based titrator which was developed for gradient flow titrations. Concentration gradients were generated electrolytically or volumetrically in small tubes. Complete titration curves can be recorded on-line and evaluated automatically. The titrator can be used with all liquid flow detectors with low axial dispersion. The titrator was evaluated for the titration of thiosulphate with electrogenerated triiodide and for the titration of ammonia with electrogenerated hypobromite after continuous gas dialytic separation of ammonia from the sample solution.