Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels

The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30.     

Electrophoretic gels of dentin matrix proteins as diffusion media for in vitro mineralization

Non-collagenous proteins of dentin and bone have important effects on mineralization which have been studied by various in vitro systems. We developed an in vitro mineralization system using electrophoretic gels as diffusion media of calcium and phosphate ions. Calcium and phosphate ions were diffused naturally or propelled by electric potential. Calcium phosphate was precipitated in the gel, and the precipitation was affected by proteins in the gel which had been separated by electrophoresis. We applied this system to analysis of non-collagenous proteins of dentin. Among the proteins, phosphophoryns promoted calcium phosphate precipitation in the natural-diffusion system. A non-collagenous protein having a molecular mass of 60 kDa inhibited precipitation. The results were different, however, in the electric-diffusion system, in which phosphophoryns had a negative effect. The present system enabled us to compare the effects of plural proteins rapidly, even using unpurified material.     

Zigzag diffusion paths in multiphase diffusion couples

The diffusion path in a multiphase diffusion couple is predicted to follow a zigzag course when the following conditions are met: the concentration differences between the diffusion couple alloys are small, the alloys contain the same phases, there is one more component in the alloys than the number of phases, and all diffusion takes place in one continuous phase that has a constant diffusivity. An analytical model for the general case of n components and p phases is given that consists of both Heaviside and error functions. The model is consistent with a proposal made by Roper and Whittle concerning “composite diffusion paths” in two-phase ternary systems.     

Lateral diffusion in an archipelago. Single-particle diffusion

Several laboratories have measured lateral diffusion of single particles on the cell surface, and these measurements may reveal an otherwise inaccessible level of submicroscopic organization of cell membranes. Pitfalls in the interpretation of these experiments are analyzed. Random walks in unobstructed systems show structure that could be interpreted as free diffusion, obstructed diffusion, directed motion, or trapping in finite domains. To interpret observed trajectories correctly, one must consider not only the trajectories themselves but also the probabilities of occurrence of various trajectories. Measures of the asymmetry of obstructed and unobstructed random walks are calculated, and probabilities are evaluated for random trajectories that resemble either directed motion or diffusion in a bounded region.     

Measurement of the ferric diffusion coefficient in agarose and gelatine gels by utilization of the evolution of a radiation induced edge as reflected in relaxation rate images

A method has been developed to determine the diffusion coefficients of ferric ions in ferrous sulphate doped gels. A radiation induced edge was created in the gel, and two spin-echo sequences were used to acquire a pair of images of the gel at different points of time. For each of these image pairs, a longitudinal relaxation rate image was derived. From profiles through these images, the standard deviations of the Gaussian functions that characterize diffusion were determined. These data provided the basis for the determination of the ferric diffusion coefficients by two different methods. Simulations indicate that the use of single spin-echo images in this procedure may in some cases lead to a significant underestimation of the diffusion coefficient. The technique was applied to different agarose and gelatine gels that were prepared, irradiated and imaged simultaneously. The results indicate that the diffusion coefficient is lower in a gelatine gel than in an agarose gel. Addition of xylenol orange to a gelatine gel lowers the diffusion coefficient from 1.45 to 0.81 mm2 h-1, at the cost of significantly lower Rl sensitivity. The addition of benzoic acid to the latter gel did not increase the Rl sensitivity.     

Freezing D2O clay gels

To obtain the T1 surface value in smectites/D2O diluted suspensions or gels, as was obtained on a monolayer deuterated clay, we freeze them. The broad Pake's doublets similar to ice doublets and with the same T1 show that we can separate frozen from unfrozen D2O. The latter exhibits a narrower line and a single T1 and is attributed to the liquid surface water layer in rapid exchange with the nearby supercooled water, the quantity of which diminishes with the lowering of the temperature depending on the gel porosity. It is possible to measure the supercooled water quantity and to correct the T1 measured values to extract the T1 surface. The value extrapolated at room temperature allows the complete clay surface area measurement. The example of a montmorillonite is given and a comparison with laponite is made.     

Migration of single-stranded DNA in polyacrylamide gels during electrophoresis

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.