Observation by Streaming Potential

Measurement of streaming current (Daly et al. 2003) and streaming potential (Etheve and Dejardin 2002; Vasina and Dejardin 2004) during adsorption can be useful to obtain information on the orientation of molecules at interfaces. The variation of streaming potential with protein interfacial concentration exhibits a change at some critical concentration for the a-chymotrypsin/mica system at pH 8.6 in 10-2M Tris (Fig. 8). The variation in zeta potential was deduced from the streaming potential, Es, as a function of time at a defined pressure differential, AP, via the classical Smoluchowski relationship (Hunter 1981), with AEs = Es - Es0, where Es0 is the asymmetry potential,

AEsnA0 , N

* AP £0£r where A0 is the electrolyte solution conductivity, n is the solution viscosity, £0 is the vacuum dielectric permittivity and £r is the relative permittivity.

The quantitative analysis of the data, however, is not simple as initially, when transport is important, the interfacial concentration is not uniform. Indeed, the Smoluchowski relationship assumes a uniform charge density along the channel walls. For another system - lysozyme/silica capillary; pH 7.4,10-2 M phosphate buffer (Etheve and Dejardin 2002) - we observed the same kind of variation and assumed that the streaming potential resulted from the mean charge density along the capillary and estimated by numerical simulations the average interfacial concentration. This did not

Fig. 8. Variation of the Z potential, relative to the initial Zo potential (-95 mV) of the bare mica, as a function of a-chymotrypsin interfacial concentration. Solution concentration Cb = 2.5 |g ml-1 in Tris 10-2 M, pH 8.6. Wall shear rate 120 s-1. The full line represents the expected variation, based on the behavior at small 6, for particles bearing only one type of charge

Fig. 8. Variation of the Z potential, relative to the initial Zo potential (-95 mV) of the bare mica, as a function of a-chymotrypsin interfacial concentration. Solution concentration Cb = 2.5 |g ml-1 in Tris 10-2 M, pH 8.6. Wall shear rate 120 s-1. The full line represents the expected variation, based on the behavior at small 6, for particles bearing only one type of charge lead to any dramatic changes in the observation of the transition. After the transition, local and average concentrations become much closer.

When comparing to similar works with random deposition of uniformly charged particles (Zembala and Adamczyk 2000) where such transition was not observed, it was concluded that the transition could be the result of the peculiar nature of the proteins. Contrary to the particles, proteins bear the two types of charge, positive and negative. Moreover, the charge distribution on the protein surface is not uniform. Let us analyze the present system along the same lines as the ones used for particles. As a first approximation, a-chymotrypsin can be assumed globally neutral because its isoelectric point is 8.2. For particles, the variation of with coverage was described as obeying a sum of an exponential function and a linear one (Zembala and Adamczyk 2000).

$0 -0 p where Z0 and Zp are the potentials of the bare surface and uniformly charged particle, respectively, in the presence of buffer. After additional control experiments, another expression was proposed (Zembala 2004):

Go Go

When the particle radius r is much larger than the Debye length k then C0 = -10.21 and Cp = 6.51. The dependence of those coefficients on rK has been established (Zembala et al. 2001). As the dimensions of a-chymotrypsin are 5.1 X 4 X 4 nm3, with r = 2-2.5 nm and k-1 = 6.3 nm, we have rK = 0.3 - 0.4, thus C0 ~ -4.5-5. As the net protein charge is almost zero, according to the model, the variation of should follow an exponential decay, exp(-C00), corresponding to the flow attenuation or substrate charge screening by the deposited neutral protein particles. We found the constant 1.8 instead of 4.5 (Fig. 8). The passage from local to mean coverage by simulations leads to the slightly lower value of 1.7. The smaller decrease in £/£0 with coverage, compared to what should be expected from the model, may have two explanations: the distribution of charges on the protein surface and/or the nonrandom adsorption (Dabros and van de Ven 1993). It is clear, however, that whatever the mechanism, proteins and homogeneously charged particles have different qualitative behaviors at high coverage. The reason is probably the protein-protein interaction associated with the mobility of proteins on the surface. As a-chymotrypsin possesses a high dipole moment (483 D; An-tosiewicz and Porschke 1989), the reasonable model of parallel dipoles at low ionic strength and low coverage, based on the ionic interaction between the mica and the protein, would lead at high coverage to a strong repulsion component between parallel dipoles, when the distance between like charges become shorter. As experiments show that the maximum interfacial concentration for proteins, and especially here for a-chymotrypsin, is generally near the full close-packed monolayer, some specific mechanism should occur.

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