Although vitamin C is essential as an antioxidant and as a cofactor in a series of enzymatic reactions, the ability for ascorbate biosynthesis was lost in humans. Thus, horticultural products and derived fruit drinks or commercial vitamin C products are considered to be important sources for the ascorbic acid intake in the human diet. These facts underline the importance of analytical methods for ascorbic acid determination in different food products.In our study two spectrophotometric and a fluorometric ascorbic acid determination methods have been compared with each other and with the so-called etalon HPLC method to find the best for small or middle sized food analytic laboratories with a sample number of up to several hundreds. As a result of our experiments we could establish that the OPDA-fluorometric method can be suggested for the determination of samples containing ascorbate at low concentrations. Unfortunately, the analytical properties of the OPDA method with spectrophotometric detection have been lagging far behind the others. The 2,2'-bipyridyl method could give a balanced performance for all tests. Furthermore, the results gained by this method are the closest to the results of the reference HPLC method in the case of fruit and vegetable samples.

The temperature profile of the normalized fluorescence resonance energy transfer efficiency is capable of monitoring the relative change of flexibility and/or conformational state of macromolecules [Biochemistry 23 (1984) 3403]. The method described earlier for one donor–one acceptor systems is extended to multiple fluorophore systems when the energy transfer occurs between either one donor–m acceptors, or n donors–one acceptor or n donors–m acceptors (where n and m are integer values). It is shown that the normalized energy transfer efficiency obtained for systems containing multiple labels is a linear combination of the normalized transfer efficiency assigned to individual donor–acceptor pairs of the system, thus its temperature profile is capable of monitoring the change of intramolecular flexibility and/or conformational state.

The activity and enantioselectivity of Candida rugosa lipase were investigated in chiral solvents, (−)-, (+)- and racemic carvone, for the resolution of 2-chloro-propionic acid with n-butanol via esterification. The activity of the enzyme studied was about 50% higher in (−)-carvone than in (+)-carvone, however the enantioselectivity was similar.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

Fluorescence techniques can be used to obtain information about biological objects in a non-destructive manner. One of these techniques is fluorescence quenching which involves a decrease in the fluorescence emission of a biological object by externally added quenchers. Quencher molecules produce two kinds of quenching: static and dynamic. Static quenching occurs due to encounter pair formation between quencher and fluorophore molecules, while dynamic quenching requires bimolecular collisions. Unless one of the mechanisms can be neglected, steady state quenching experiments cannot provide information on the contributions of the two processes. However, time-resolved experiments are sensitive only to the dynamic process, and thus provide selective information about the relative motion of the quencher and fluorophore. Since the two quenching events are controlled by different physicochemical parameters, it is necessary to resolve them. In this paper, we describe a steady state method to resolve the static and dynamic quenching constants (rather than time-resolved techniques). Our method is based on the simultaneous determination of the fluorescence intensity and emission anisotropy data and can be regarded as the further development of quenching-resolved emission anisotropy (QREA). Since the steady state anisotropy and fluorescence lifetime are inversely related, by determining the steady state fluorescence anisotrophy, changes in the fluorescence lifetime (and hence the dynamic quenching process) can be monitored (if other parameters influencing the anisotropy remain constant). We present a theoretical description of the method, computer simulations testing its accuracy and results of model experiments with pyridoxamine-phosphate-labelled lysozyme and acrylamide. By changing the external viscosity, we obtained data on the theoretical inverse relationship between the dynamic quenching constant and viscosity. The application conditions are also discussed.

The parameters characterizing the quenching of fluorescence emitted by the coenzyme (pyridoxal-5′-phosphate) of phosphorylase b (EC 2.4.1.1) by anions are good indicators of conformational/dynamic changes at the active center. Reinvestigation of this quenching process resulted in a non-linear Stern-Volmer plot. This non-linearity is described by a simple kinetic model which assumes two parallel processes, one represented by bound and the second by free quencher molecules. Analysis of separate parts of the nonlinear Stern-Volmer plot results in the values of rate constants for the bound and free quencher molecules as well as the value of dissociation constant of the anions.

Membrane-related events can be investigated when the fluorescence of an intramembrane fluorophore is quenched by molecules that are dissolved in lipid phase. In this case the bimolecular quenching constant characterises the relative transport rate of the fluorophore and quencher molecules in the membrane interior and thereby it is related to the dynamics or structure of the membrane. Unlike classic quenching experiments, the crucial point in such studies is that the concentration of the quencher in the lipid phase differs from that in the bulk. As a consequence, it is usually described by different models, or regarded as the total concentration added. Here a simple fluorometric study is presented for distinguishing between the solvation mechanisms (partition or binding) of quencher molecules in membrane phase.

Subcritical micellar concentrations (sub-CMC) of Brij-series detergents alter ion movements between human erythrocytes and their environment when metabolism has been slowed down by incubation at zero degrees centigrade. The effect of nonhemolytic concentrations of detergents on the erythrocyte K+ and Na+ movements is described. Results indicate a significant difference in monovalent cation movements, depending on the number of hydrophilic polyoxyethylene units (n). There is an increasing loss of K+ and gain of Na+ as n increases from 4 to 20. Where n > or = 21, ion movements are not significantly different from those found in erythrocytes not exposed to detergents. The carbon chain length of the detergent fatty acid residue (10-18 carbons) appears to be relatively unimportant, but detergents with unsaturated (oleic acid) hydrophobic regions potentiate K+ release and Na+ uptake when compared to the corresponding saturated fatty acid (stearic acid). The erythrocyte stabilizing effect of detergents against hypo-osmotic shock correlates well with the increase of monovalent ion traffic and the mobility of membrane lipids revealed by fluorescence anisotropy measurements.