Surface exclusion and molecular mobility may explain Vroman effects in protein adsorption

Data on protein adsorption usually show that for increasing surface coverage the adsorption velocity decreases much faster than linearly. This contrasts to the classical Langmuir model with an adsorption velocity proportional to the number of unoccupied binding sites. It has been shown that this non-linearity may explain phenomena like transient adsorption of different proteins from a protein mixture or dilution-dependent changes in binding properties, collectively called Vroman effects. However, the molecular mechanisms explaining this non-linear behavior remain to be established. A Monte Carlo simulation model is presented that incorporates steric hindrance, lateral mobility and mutual interactions of adsorbed molecules. Experimental data on the adsorption kinetics of prothrombin and annexin V, a recently discovered anticoagulant protein, at phospholipid bilayers are analyzed with this model. A major conclusion is that the steep decline in adsorption rates for increasing surface coverage can be explained, without assuming repulsive forces between adsorbed molecules, as a surface exclusion effect combined with lateral mobility of adsorbed molecules. The fact that annexin V shows this effect to a much lesser degree than prothrombin is tentatively explained by clustering of adsorbed annexin V molecules. A qualitative effect of lateral mobility on the adsorption characteristics, predicted by the model, is confirmed in experiments in which the fluidity of the bilayers was manipulated

Annexin V perturbs or stabilises phospholipid membranes in a calcium-dependent manner

AbstractThe potency of annexin V to transport Ca2+ ions across phospholipid membranes was investigated, using large unilamellar phospholipid vesicles loaded with the Ca2+ indicator fura-2. It was demonstrated that annexin V leaves the vesicle membranes intact when added in the presence of 1 mM Ca2+. However, if the vesicles were first incubated with annexin V in the absence of Ca2+, subsequent addition of Ca2+ produced a fluorescence signal due to binding of Ca2+ to fura-2. Centrifugation of the vesicle suspension immediately thereafter showed that this signal originated from the supernatant and not from the sedimented vesicles. Our results show that annexin V causes loss of vesicle integrity in the absence of Ca2+, and leakage of trapped fura-2, rather than inward Ca2+ transport. Bovine serum albumin or Ca2+ concentrations higher than 2.5 mM also caused such fura-2 leakage. Apparently, calcium-dependent binding of annexin V to the membrane prevents aspecific membrane damage caused by this protein

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20Va dioleoylphosphatidylserine (DOPS) and 80Vo dioleoylphosphatidylcholine (DOPC) phospho-lipid mixture. For prothrombin, a dissociation constant Kd:140+27 nM (mean*S.E.) and maximal surface concentration fL".: (8.9 + 0.8) ' 10- 3 mole of protein per mole of lipid, were obtained. For factor Xa, these values were K d: 49.6 + 6.3 nM and 1-u *:Q3.0 t 1.4) ' 10-3 mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature. r2

Cumulative Troponin T Release after Acute Myocardial Infarction. Influence of Reperfusion

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