Interaction of dystrophin fragments with model membranes.

The interaction with membrane lipids of recombinant fragments of human dystrophin, corresponding to a single structural repeating unit of the rod domain, was examined. Surface plasmon resonance, constant-pressure isotherms in a Langmuir surface film balance, and interfacial rheology were used to observe binding of the polypeptides and its effects on the properties of the lipid film. Modification of the monolayer properties was found to depend on the presence of phosphatidylserine in the lipid mixture and on the native tertiary fold of the polypeptide; thus a fragment with the minimum chain length required for folding (117 residues) or longer caused a contraction of the surface area at constant pressure, whereas fragments of 116 residues or less had no effect. The full extent of contraction was reached at a surface concentration of lipid corresponding to an average area of about 42 A2 per lipid molecule. A dystrophin fragment with the native, folded conformation induced a large increase in surface shear viscosity of the lipid film, whereas an unfolded fragment had no effect. Within a wide range of applied shear, the shear viscosity remained Newtonian. Binding of liposomes to immobilized dystrophin fragments could be observed by surface plasmon resonance and was again related to the conformational state of the polypeptide and the presence of phosphatidylserine in the liposomes. Our results render it likely that intact dystrophin interacts directly and strongly with the sarcolemmal lipid bilayer and grossly modifies its material properties.

Flash freezing of erythrocyte suspensions.

Freezing whole blood in bulk usually results in severe cellular destruction through the action of ice crystals and osmotic effects in the freezing liquid. The potential of flash freezing blood aerosols onto a liquid nitrogen surface as a means of inhibiting cellular damage was studied in this work. Three commercial spraying devices were employed to spray-freeze either whole blood or concentrated erythrocyte suspensions, using hydroxyethyl starch (HES) as a cryoprotectant. The integrity and viability of the processed cells were assessed by measuring gross rheological properties and the extent of hemolysis. Cells were found to be susceptible to the very high shear stresses imposed by some of the spraying devices. Bulk freezing of blood, even in the presence of cryoprotectant, resulted in complete cellular destruction. Whereas flash freezing was capable of substantially reducing the level of hemolysis to 12.6% and preserving the cellular deformability.

Experimental phase changes at the mica-silver interface illustrate the experimental accuracy of the central film thickness in a symmetrical three-layer interferometer.

Experimentally measured phase changes of light on reflection at the mica-silver interface are reexamined and found to be in agreement with those calculated using modern optical constants. Phase changes on reflection at a dielectric-silver interface can therefore be calculated using the well-known analytical (cf. empirical) expressions and the optical constants, provided the refractive index of the dielectric is known or measured and the silver films are prepared in a similar manner. This discussion is relevant to measurements obtained from the surface forces apparatus. When the surface separation is calculated by Airy's method, we show that the phase changes on reflection at the dielectric-silver interface at the reference wavelengths are either explicitly or implicitly accounted for in all the expressions. We also show that the surface forces technique (spectrometer resolution, ~32 Å mm(-1)) is inaccurate for measuring the thickness of very thin aqueous films (<10 Å) and that for all practical purposes the central film thickness has to be >50 Å to achieve a resolution of 1 Å.

Forces between proteins and model polypeptides adsorbed on mica surfaces.

The forces of interaction between proteins adsorbed onto mica have been measured as a function of the distance of separation between the two mica surfaces in aqueous solutions. The results for three proteins, myelin basic protein, concanavalin A and cytochrome c, are presented together with the results for a model basic protein, poly(L-lysine). With the exception of cytochrome c at large separations, the forces of interaction are due to charges on the protein surfaces and may be fitted closely to theoretical predictions. For cytochrome c, however, no long-range electrical repulsion is observed, indicating that the negatively charged mica surface has been neutralised by the adsorption of the positively charged protein. At short surface separations, an attraction between the protein surfaces was noted. For concanavalin A, a weak attraction was observed in the presence of calcium and manganese ions only. For poly(L-lysine) and cytochrome c the attraction can be explained simply in terms of van der Waals interactions between the proteins. However, for myelin basic protein the observed attraction was an order of magnitude larger than that predicted by van der Waals theory. We believe that this additional attraction may be due to hydrophobic interactions between the adsorbed myelin basic protein molecules.