Biopolymers

BIOPOLYMERS

Many biological macromolecules, such as proteins and polysaccharides, are charged polymers. Upon combining proteins and polysaccharides in solution, a vast range of interesting phase behavior, which is strongly dependent upon ionic strength, relative biopolymer concentration, pH, temperature, and functional groups of the biopolymers, can occur.

The polysaccharide-protein mixture we are interested in is hyaluronic acid and the globular plasma proteins (which consist primarily of serum albumin and gamma-globulins). These macromolecules are the main constituents of synovial fluid, found in mammalian joints. We are studying the lubrication mechanism through rheological measurements.

Figure 1 - Possible mechanisms by which both a stable one phase system, and also spontaneous biphasic demixing can occur.

Mixtures of proteins and polysaccharides are stable in one phase as co-soluble mixtures, or soluble complexes. Spontaneous demixing will occur when the protein and polysaccharide bind to each other into an insoluble complex, or when the two biopolymers are incompatible and partition into the two different phases. Demixing can result in liquid-liquid, liquid-gel, or liquid-solid (as in the case of precipitation) systems.

Rheology can be used to probe this phase behavior. As viscosity is very sensitive to molecular weight, h~M^3.4 above the entanglement molecular weight), complex formation, which effectively raises the molecular weight of the molecules, will result in a drastic rise in the viscosity. However, the rheological properties of a species also scale with its concentration. Therefore if microphase separation leads to regions of higher concentration, the viscosity of these regions will also go up.

Figure 2 - Time dependent viscosity of a mixture of hyaluronic acid, 3mg/mL, and the plasma proteins bovine serum albumin, 11mg/mL and gamma globulins 7mg/mL (red), and the viscosity of synovial fluid from a bovine stifle joint (black). It is evident that a structural change is occurring over time, and this may be facilitated by applying low shear rates to the fluid (dg/dt=0.02-1.0s^-1).

Alone, the rheological techniques cannot explain which of these changes are occurring. Thus, other techniques must be employed to determine the details of the phase behavior. Scattering techniques can be used to determine the size (or the molecular weight) of the species in solution. Since the increase in the size of the polymers or an increase in the molecular weight is due to complex formation or protein aggregation, this can be easily determined by scattering. Binding (soluble or insoluble complex formation) can be discerned by methods sensitive to the chemical potential of the solution, such as osmotic pressure or equilibrium dialysis.

BIOPOLYMERS

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