Particle diffusion in crowded media was studied through Monte Carlo simulations in 3D obstructed lattices. Three particular aspects affecting the diffusion, not extensively treated in a three-dimensional geometry, were analysed: the relative particle-obstacle size, the relative particle-obstacle mobility and the way of having the obstacles distributed in the simulation space (randomly or uniformly). The results are interpreted in terms of the parameters that characterize the time dependence of the diffusion coefficient: the anomalous diffusion exponent (α), the crossover time from anomalous to normal diffusion regimes (τ) and the long time diffusion coefficient (D*). Simulation results indicate that there are a more anomalous diffusion (smaller α) and a lower long time diffusion coefficient (D*) when obstacle concentration increases, and that, for a given total excluded volume and immobile obstacles, the anomalous diffusion effect is less important for bigger size obstacles. However, for the case of mobile obstacles, this size effect is inverted yielding values that are in qualitatively good agreement with in vitro experiments of protein diffusion in crowded media. These results underline that the pattern of the spatial partitioning of the obstacle excluded volume is a factor to be considered together with the value of the excluded volume itself.

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.

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.

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, v_max and K_m. 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.

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 α-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, α-chymotrypsin shows anomalous diffusion (α < 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.