Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.

Two-photon fluorescence cross-correlation spectroscopy.

Dual-color cross-correlation spectroscopy is a special kind of fluctuation analysis which selectively probes the formation or deletion of linkages between two different fluorescently labeled molecules at extremely low concentrations. Two-photon excitation can, under certain circumstances, significantly simplify this method if different probe molecules with distinct emission properties are accessible by a common IR excitation wavelength.

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis.

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

The influence of pCO2 on the rate of ammonia formation in blood.

The influence of the sample pCO2 on the rate of ammonia formation was studied with gas equilibrated blood samples, using different gas mixtures for the equilibration. The rate of increase in plasma ammonia concentration at a mean pCO2 of 62 mm Hg = 8.2 kPa (mean pH = 7.282) was significantly lower than at 36 mm Hg = 4.8 kPa (pH = 7.438). In CO2-depleted blood (pH > 8) ammonia formation was strongly accelerated. This was reversible by readjusting the pH to 7.4 by addition of Tris-HCl solution. In stoppered containers with or without enclosed atmospheric air, a decrease of blood pCO2 or an increase of pH values was not observed during storage over 15 minutes at 0 or 20 degrees C. Although this study confirms that the pCO2 (or rather the pH) is an important analytical influence quantity in the determination of plasma ammonia, strictly anaerobic processing of the blood samples is not necessary; the usual technique of transporting and preprocessing blood samples in partially filled and stoppered containers appears to be adequate. Mainly due to the deamination of intracellular AMP (1), the ammonia1) concentration in blood increases continuously after sampling. Rates of increase in plasma ammonia concentration have recently been investigated thoroughly with blood samples from healthy probands to define the maximum delay between sampling and separation of the blood cells that can be tolerated if the in vivo existing plasma ammonia concentration has to be measured (2). Strictly speaking, the guidelines for handling the blood samples (2) apply to blood stored under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Which is the appropriate coenzyme for the measurement of ammonia with glutamate dehydrogenase?

The determination of ammonia in plasma, using glutamate dehydrogenase, is complicated by non-specific oxidation of the coenzyme, promoted by components of the sample matrix. Measurements performed with appropriate plasma blanks show that 2'-phosphorylated coenzymes (NADPH, deamino-NADPH) are much less oxidized than NADH. By adding lactate dehydrogenase, NADH oxidation by endogenous pyruvate can be completed within a short time. Considerable consumption of coenzyme occurs, however, and endogenous L-alanine aminotransferase also represents a possible source of interference. The results of ammonia determinations using deamino-NADPH (y) or NADPH (x) were identical (a = 0.0 mumol/l, b = 1.00; r = 0.996, n = 62). With NADH as the coenzyme, the method displays adequate specificity only at high sample dilution, e.g. in the measurement of urea after conversion to ammonia.

Temperature-dependent matrix effect in the direct enzymatic measurement of blood glucose.

The influence of the high-molecular-mass sample matrix in the direct enzymatic measurement of glucose in haemolysate was investigated by a comparison study using ultrafiltered haemolysate for reference. Haemolysate was obtained by 1:21 dilution of whole blood with a solution of digitonin and maleinimide. It was shown that at low protein concentration glucose distributes in a 1:1 ratio during ultrafiltration. With a hexokinase/glucose-6-phosphate dehydrogenase procedure excellent agreement was found between values measured in haemolysate (y) and ultrafiltrates (x), when incubation was performed at 25 degrees C (a = 0.047 mmol/l; b = 0.99; r = 0.999, n = 37); at 37 degrees C, however, the same procedure resulted in a non-tolerable systematic deviation in the direct analysis of haemolysate (a = -0.426 mmol/l; b = 1.00, r = 0.997, n = 37). The precision of measurements in haemolysate and ultrafiltrate was similar (CV 1.0-1.2%). Since stable reference material with an appropriate matrix is not available, it is important to evaluate haemolysate procedures carefully by comparison studies with patient samples. For reduction of experimental error in such studies we recommend the use of ultrafiltered haemolysate, since this can be analysed side by side with haemolysate in the same run.

Serum ultrafiltration for the elimination of endogenous interfering substances in creatinine determination.

Serum, at neutral pH, was submitted to a simple filtration, using centrifugation in the disposable Centrisart. The ultrafiltrate was similar to serum in its creatinine content but was virtually free from proteins, including protein-bound bilirubin, haemoglobin and lipoproteins. The creatinine concentrations of anicteric serum specimens and the corresponding ultrafiltrates as determined with Jaffé and enzymic procedures show a high correlation and are convertible. With icteric sera the negative interference effect of bilirubin in a particular analytical procedure can be quantified using ultrafiltrate as the reference. It is suggested that ultrafiltration is useful in selected cases for eliminating elevated concentrations of bilirubin, haemoglobin and turbidity, which would interfere in the direct creatinine determination. Relative to the continuous flow methods with dialysis of the analyte, direct methods for creatinine are more susceptible to interference by endogenous factors like hyperbilirubinaemia, hypertriglyceridaemia and haemolysis (1). The negative interference by bilirubin is of special importance, since it interferes in some modifications of the kinetic Jaffé method (2) and in the chromogenic enzymatic method (3). As a simple alternative, we evaluated the use of serum ultrafiltrate for the accurate determination of creatinine by the Jaffé and enzymatic methods, free from interfering by the high-molecular serum matrix and compounds bound to it.