Comparison of the pH dependent calculated and known molecular weight values for human and bovine insulin.įormulation additives, such as soaps and salts, can have a pronounced influence on the surface charge density of the protein and the solution ionic strength. Size distributions for human and bovine insulin at pH 7 and pH 2, indicating a pH dependent change in quaternary structure. At pH 7, the measured diameters are consistent with the known hexameric forms of the proteins at physiological pH.įigure 2. At pH 2, the measured diameters for both proteins (see Table 1) are consistent with dimeric quaternary structures, where the molecular weight is estimated from empirically determined size vs. For example Figure 2 shows the measured size distributions for human and bovine insulin at pH 2 and pH 7. The precision of dynamic light scattering measurements is sufficient to distinguish changes in protein quaternary structure. The quaternary structure or ordered self-association state of a protein can be influenced by solution properties such as the pH and the ionic strength. Thermal scan for bovine haemoglobin in 0.13 M phosphate buffered saline, indicating a melting point of 45.5 C. Figure 1 shows a temperature scan for bovine haemoglobin, and clearly indicates a sharp increase in both the size and scattering intensity at the melting point of 45.5☌.įigure 1. Because of the molecular weight dependence of the scattering intensity, this non-specific aggregation of denatured proteins is easily monitored with light scattering instrumentation. This entropically unfavourable state is soon replaced however, with one wherein the hydrophobic residues on one protein chain associate with those on another protein chain. When a protein denatures, the hydrophobic residues buried within the interior of the folded structure are exposed to the solvent. The temperature at which this denaturation occurs is defined as the protein melting point. As energy is added to the system via an increase in temperature, the stabilizing forces can be disrupted, allowing the protein to unfold or denature. The structure of a protein is stabilized by a large number of hydrogen bonds, hydrophobic interactions, and Van der Waal forces, each of which contributes a small degree of stability to the overall structure. Today's generation of light scattering instrumentation includes highly stable lasers, fiber optics, high speed correlators, and single photon counting detectors that facilitate the measurement of protein samples across a range of size and concentration that has never before been achievable. As such, dynamic and static light scattering techniques are very sensitive to the onset of protein aggregation arising from subtle changes in the solution conditions. The scattering intensity of a small molecule is proportional to the square of the molecular weight. Light Scattering as a Characterization Tool Light scattering is a non-invasive technique that has received wide acceptance in the area of protein and formulation characterization. Because of the sensitivity of the protein to solution changes, invasive characterization techniques can be problematic. The stability of a protein formulation to a variety of solution perturbations is critical to its success as a pharmaceutical product. Sponsored by Malvern Panalytical Apr 20 2005
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