Flowing protein solution, kinetics

Protein Adsorption. The development of medical implant polymers has stimulated interest in the use of ATR techniques for monitoring the kinetics of adsorption of proteins involved in thrombogenesis onto polymer surfaces. Such studies employ optical accessories in which an aqueous protein solution (93) or even ex - vivo whole blood (94-%) can be flowed over the surface of the internal reflection element (IRE), which may be coated with a thin layer of the experimental polymer. Modem FT-IR spectrometers are rapid - scanning devices, and hence spectra of the protein layer adsorbed onto the IRE can be computed from a series of inteiferograms recorded continuously in time, yielding ah effective time resolution of as little as 0.8 s early in the kinetic runs. Such capability is important because of the rapid changes in the composition of the adsorbed protein layers which can occur in the first several minutes (97). 

M 35] [protocol see [119]] A protein conformation kinetic study of the small protein ubiquitin was performed both in the continuous and in a stopped-flow mode at low reactant consumption [119], The bifurcation mixer was used prior to an IR flow cell for data monitoring. The change of conformation from native to the A-state was followed when adding methanol under low pH conditions to the protein solution. In the continuous mode, long data acquisition could be made and the reaction time was determined by the flow rate and the volume interconnecting zone between the mixer and IR flow cell, which was small, but not negligible. In the stopped-flow mode, the reaction time resolved was dependent on the time resolution of the FTIR instrument. 

Application of a quantity of solution, in this case protein solution, to a column filled with appropriate substrate, leads to an exchange between the solute and the surface, and movement with the solvent front such that the emerging solute profile contains complete information concerning the kinetics of adsorption/desorption. Apart from the interaction of solute with surface, concurrent three-dimensional diffusion often occurs, and perhaps occlusion, if the substrate is porous. Nevertheless, the option of deducing interfacial kinetics from the elution profile in a flowing system has a certain elegance and relevance to the performance of blood plasma proteins in vivo. 

Aside from the relative position of the profile, the shape of the effluent profile contains information concerning the kinetics of the adsorption process. All concentrations of protein from zero to cQ are brought into contact with the column surface as the protein solution flows through the column, as a function of the position of the profile, and therefore as a function of time. Working with small molecules, previous researchers have shown that compounds exhibiting Langmuir isotherms produce sharp fronts, and diffuse tails, if pure solvent is used to desorb the column (21,22). However, Equation 7 shows that both diffusional and adsorption effects can alter the shape of the effluent profile. The former effect includes both normal molecular diffusion, and also diffusion due to flow properties in the column (eddy diffusion), which broadens (decreases the slope) the affluent profiles. To examine the adsorption processes, apart from the diffusional effects, the following technique can be applied. 

Protein Adsorption and Desorption Rates and Kinetics. The TIRF flow cell was designed to investigate protein adsorption under well-defined hydrodynamic conditions. Therefore, the adsorption process in this apparatus can be described by a mathematical convection-diffusion model (17). The rate of protein adsorption is determined by both transport of protein to the surface and intrinsic kinetics of adsorption at the surface. In general, where transport and kinetics are comparable, the model must be solved numerically to yield protein adsorption kinetics. The solution can be simplified in two limiting cases 1) In the kinetic limit, the initial rate of protein adsorption is equal to the intrinsic kinetic adsorption rate. 2) In the transport limit, the initial protein adsorption rate, as predicted by Ldveque s analysis (23), is proportional to the wall shear rate raised to the 1/3 power. In the transport-limited adsorption case, intrinsic protein adsorption kinetics are unobservable. 

The energetics of protein association can be studied by a variety of experimental techniques,17 each of which permits measurements of equilibrium or kinetic values in a certain range. Widely used techniques include isothermal titration calorimetry, surface plasmon resonance measurement, stopped flow kinetics, optical spectroscopy, MS, and analytical ultracentrifugation. The techniques differ in their requirements (e.g., amount of protein, labeling with fluorophores, attachment to sensor surfaces, and the environment provided by the experimental set up) and therefore in their applicability to individual cases. Different techniques can also give quite different values for what might be expected to be the same quantity. For example, association rates measured by surface plasmon resonance, with one protein immobilized on a surface, are usually different from those measured for the two proteins in solution and under otherwise similar conditions. 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

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