Mária Punyiczki

Heat Shock and Apoptosis: The Two Defense Systems of the Organism May Have Overlapping Molecular Elements

Stress response and apoptosis are two interrelated end points of the defense systems of living organisms. The molecular elements of the two have strong influences on each other. Ceramide formation and signal-induced phosphorylation cascades may be critical in determining the final fate of cells exposed to environmental changes.

Plasminogen activator activity and plasminogen independent amidolytic activity in tear fluid from healthy persons and patients with anterior segment inflammation

Plasminogen activator activity and plasminogen independent amidolytic activity were measured in human tears by a spectrophotometric method using human plasminogen and chromogenic peptide substrate S-2251. This assay is sensitive predominantly to urokinase-like plasminogen activator. Tears were collected with glass capillaries. The activator activity in normal tears was found to be low, 0.06 +/- 0.04 (SD) IU/ml. Elevated levels were measured in the tears of patients with various types of conjunctival and corneal disorders. The affected epithelial cells of the cornea and conjunctiva were suggested to be responsible for the elevated activity. Plasminogen independent amidolytic activity was usually very low except in cases of increased permeability of the conjunctival blood vessels. The procedure is recommended as a useful tool for the study of the pathological changes in the epithelial cells of the cornea and conjunctiva.

Interaction of acrylamide with proteins in the concentration range used for fluorescence quenching studies

Abstract 14 C labelled acrylamide was synthesized and used in equilibrium dialysis measurements to study the binding of acrylamide to the proteins: human serum albumin (HSA), ovalbumin and cod paryalbumin III. Our intent was to determine whether binding takes place in the concentration range that is used for the study of fluorescence quenching by acrylamide. In contrast to previously published reports, we found that all the proteins investigated did bind acrylamide. The affinity of this interaction, when interpreted in terms of multiple independent binding equilibria, was very low, K ass ⋍ 0.5–2 M −1 ; however, due to multiple binding sites, a considerable amount of acrylamide is to be found in the protein phase at concentrations used for quenching experiments. The number of binding sites seems to vary with the protein. A pH 7.0 the binding to the 69 kD HSA corresponds to 14 ± 8 sites, the binding to the 45 kD ovalbumin corresponds to 40 ± 25 sites, whereas for the 11 kD cod parvalbumin the binding corresponds to only a few sites. The binding is very sensitive to pH and to the presence of cosolvents such as glycerol. At pH 5.2, close to the isoionic point, the number of binding sites for acrylamide on HSA increases to > 100. The very weak binding justifies an alternative description of the phenomena as a distribution equilibrium between two phases. In such a model we see that the formal concentration of acrylamide in the protein volume is in some cases higher than in solution ( K eq = 1.2 for HSA at pH 5.2). These findings suggest that any model describing the quenching of fluorescence in proteins by acrylamide has to account for the presence of two pools of acrylamide and consequently for the presence of multiple modes of quenching.

Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence

Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.

Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism

The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.