Isabel Pastor

Effect of crowding by dextrans on the hydrolysis of N-Succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin

Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in nonspecific interactions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin, by adding dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A v(max) decay and a K(m) increase were obtained when the dextran concentration in the sample was increased. The increase in K(m) can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in v(max) could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media. As far as we know, this is the first reported experiment on the crowding effect in an enzymatic reaction with a mixed inhibition by product.

Diffusion of alpha-Chymotrypsin in solution-crowded media. A fluorescence recovery after photobleaching study

Fluorescence recovery after photobleaching (FRAP) is one of the most powerful and used techniques to study diffusion processes of macromolecules in membranes or in bulk. Here, we study the diffusion of alpha-chymotrypsin in different crowded (Dextran) in vitro solutions using a confocal laser scanning microscope. In the considered experimental conditions, confocal FRAP images could be analyzed applying the uniform circular disk approximation described for a nonscanning microscope generalized to take into account anomalous diffusion. Considering the slow diffusion of macromolecules in crowded media, we compare the fitting of confocal FRAP curves analyzed with the equations provided by the Gaussian and the uniform circular disk profile models for nonscanning microscopes. As the fitted parameter variation with the size and concentration of crowders is qualitatively similar for both models, the use of the uniform circular disk or the Gaussian model is justified for these experiments. Moreover, in our experimental conditions, alpha-chymotrypsin shows anomalous diffusion (alpha < 1), depending on the size and concentration of Dextran molecules, until a high concentration and high size of crowding agent are achieved. This result indicates a range of validity of the idealized fitting expressions used, beyond which other physical phenomena must be considered.

Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

Effect of crowding by Dextrans in enzymatic reactions

The interior of the living cell is highly concentrated and structured with molecules that have different shapes and sizes. Almost all experimental biochemical data have been obtained working in dilute solutions, situations which do not reflect the in vivo conditions. The consequences of such crowding upon enzymatic reactions remain unclear. In this paper, we have studied and compared the initial velocity of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by alpha-chymotrypsin, the oxidation of ABTS by H2O2 catalyzed by HRP and the oxidation of NADH in presence of pyruvate catalyzed by LDH. These reactions were chosen as model enzymatic processes occurring in different in vitro crowded media. The systems crowding has been built by introducing Dextran of several concentrations and sizes. Our results indicate that the volume occupied by the crowding agent, but not its size, plays an important role on the initial velocity of reactions involving tiny enzymes. However, the enzyme size is another important factor influencing the velocity of the reactions of large enzymes occurring in Dextran crowded media. In this situation, the reaction initial velocity depends on both occupied volume and dimension of the crowding agent that is present in the reaction media.

Immobilization of a trienzymatic system in a sol-gel matrix : A new fluorescent biosensor for xanthine

In this work we report the development of a highly sensitive fluorescent multienzymatic biosensor for quantitative xanthine detection. This biosensor is built by the simultaneous encapsulation of three enzymes, xanthine oxidase, superoxide dismutase and peroxidase, in a single sol-gel matrix coupled to the Amplex Red probe. The sol-gel chemistry yields a porous, optically transparent matrix that retains the natural conformation and the reactivity of the three co-immobilized proteins. Xanthine determination is based on a sequence of reactions, namely catalytic oxidation of xanthine to uric acid and superoxide radical, and subsequent catalytic dismutation of the radical, resulting in the formation of hydrogen peroxide, which reacts stoichiometrically with non-fluorescent Amplex Red to produce highly fluorescent resorufin. The optimal operational conditions for the biosensor were investigated. Linearity was observed for xanthine concentrations up to 3.5 microM, with a detection limit of 20 nM, which largely improved the sensitivity of the current xanthine biosensors. The developed biosensor is reusable and remains stable for 2 weeks under adequate storage conditions.

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ∼15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (≤1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail.

Model-independent link between the macroscopic and microscopic descriptions of multidentate macromolecular binding: Relationship between stepwise, intrinsic, and microscopic equilibrium constants

The binding of ions or other small molecules to macromolecules and surfaces can be macroscopically characterized by means of the stepwise (or stoichiometric) equilibrium constants, which can be obtained experimentally from coverage versus concentration data. The present work presents a novel, simple, and direct interpretation of the stepwise constants in terms of the microscopic, site-specific, stability constants. This formalism can be applied to the most general case, including the heterogeneity of the sites, interactions among them, multicomponent adsorption, and so forth, and, in particular, to chelate complexation. We show that the stepwise equilibrium constants can be expressed as a product of two factors, (i) the average number of free potential sites (per bound ion) of the microscopic species to be complexed (stoichiometric factor) and (ii) the average of the microscopic stability constants of their free potential sites. The latter factor generalizes the concept of the intrinsic equilibrium constant to systems with chelate complexation and reduces to the standard definition for monodentate binding. However, in the case of heterogeneous multidentate complexation, the stoichiometric factor cannot be known a priori, so that the finding of the intrinsic constants is not trivial. One option is to approximate the stoichiometric factor by the value that would correspond to identical active centers. We investigate the accuracy of this assumption by comparing the resulting approximate intrinsic constants to those obtained by Monte Carlo simulation of several binding models. For the cases investigated, it is found that the assumption is quite accurate when no correlated structures (typical of short-range interactions) are formed along the chain. For adsorption of particles attached to a large number of active centers, the formalism presented here leads to the Widom particle insertion method.

Monte Carlo simulations of enzymatic reactions in crowded media. Effect of the enzyme-obstacle relative size

We perform Monte Carlo simulations in three-dimensional (3D) lattice in order to study diffusion-controlled and mixed activation-diffusion reactions following an irreversible Michaelis-Menten scheme in crowded media. The simulation data reveal the rate coefficient dependence on time for diffusion-controlled bimolecular reactions developing in three-dimensional media with obstacles, as predicted by fractal kinetics approach. For the cases of mixed activation-diffusion reactions, the fractality of the reaction decreases as the activation control increases. We propose a modified form of the Zipf-Mandelbrot equation to describe the time dependence of the rate coefficient, k(t)=k0(1+t/τ)(-)(h). This equation provides a good description of the fractal regime and it may be split into two terms: one that corresponds to the initial rate constant (k0) and the other one correlated with the kinetics fractality. Additionally, the proposed equation contains and links two limit expressions corresponding to short and large periods of time: k1=k0 (for t≪τ) that relates to classical kinetics and the well-known Kopelmans equation k∼t(-)(h) (for t≫τ) associated to fractal kinetics. The τ parameter has the meaning of a crossover time between these two limiting behaviours. The value of k0 is mainly dependent on the excluded volume and the enzyme-obstacle relative size. This dependence can be explained in terms of the radius of an average confined volume that every enzyme molecule feels, and correlates very well with the crossover length obtained in previous studies of enzyme diffusion in crowding media.

Multienzymatic system immobilization in sol-gel slides: Fluorescent superoxide biosensors development

One of the potential areas of research in the development of biosensors is the production of analytical devices based on the use of immobilized multienzymatic systems. In this work, we report the development of three analytical systems for superoxide radical detection using sol-gel technology to immobilize enzyme systems. These systems are based on the connected reactions of three enzymes (xanthine oxidase, superoxide dismutase and horseradish peroxidase) coupled to the probe Amplex red. The difference between these three systems lies in the immobilization of two or three enzymes into a single or in different sol-gel slides. We check the potential use of each designed systems to quantify superoxide radical and potential evaluation of radical scavenging properties of several antioxidant compounds.

Influence of macromolecular crowding on the oxidation of ABTS by hydrogen peroxide catalyzed by HRP

Influence of Macromolecular Crowding on the Oxidation of ABTS by Hydrogen Peroxide Catalyzed by HRP The interior of the living cell is highly concentrated and structured with molecules having different shapes and sizes. However, almost all experimental biochemical data have been obtained working in dilute solutions that do not reflect in vivo conditions.