Protein characterization

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Chapter: Pharmaceutical Drugs and Dosage: Protein and peptide drug delivery

Biophysical characterization of Protein , Physicochemical characterization of Protein


Protein characterization

Biophysical characterization of Protein 

Therapeutic applications of proteins require an understanding of fully elu-cidated structure, pharmacology, and mechanism of action. In addition, protein behavior in solution and the impact of chemical properties and components of solutions on the physical properties of solutions (termed bio-physical characterization) need to be well defined. Biophysical character-ization of proteins includes the determination of size, shape, and solution properties of proteins through direct and indirect techniques that include the following:

·      Hydrodynamic protein size measurement by analytical ultracentri-fugation, gel filtration chromatography, gel electrophoresis, and/or viscometry.

·       Thermodynamic methods such as microcalorimetry and surface plasma resonance can help delineate the state of protein association and interactions with other molecules in solution.

·           Particulate formation by protein self-association or interaction with other components in solution by dynamic light scattering (DLS).

·           Spectroscopic methods such as circular dichroism (CD) and thermal melt fluorescence spectroscopy can help determine the stability of protein conformation in solution.


Physicochemical characterization of Protein 

Solubility

Under physiological conditions, solubility of proteins can vary from the very soluble to the virtually insoluble. Water solubility of a protein requires interactions, such as hydrogen bonding and electrostatic inter-actions, of protein surface with the aqueous medium. The hydrophilic interactions, which are stronger and predominant in aqueous conditions, are enhanced by the ionization of functional groups on proteins such as amines and carboxylates. Ionization of these functional groups is pH dependent. Thus, the solubility of proteins and peptides is dependent on the pH of the solution.

The overall charge on a protein can be either positive or negative, depend-ing on the ionization status of all of its functional groups. A protein is usu-ally positively charged at a low pH and negatively charged at a high pH. Protein solubility increases as the pH of the solution moves away from the isoelectric point (IEP) (Figure 25.7), which is the pH at which the mol-ecule is ionized but has a net zero charge and does not migrate in an elec-tric field (determined by gel electrophoresis). The presence of both positive and negative charges on the protein at its IEP leads to a greater tendency for self-association. As the net charge on the protein changes in any one direction (positive or negative) with a change in solution pH, the affin-ity of the protein for the aqueous environment increases and the protein molecules also exert greater electrostatic repulsion among each other, thus preventing them from self-associating. This increases their aqueous solubil-ity. However, extremes of pH can cause protein unfolding with the expo-sure of hydrophobic groups and protein self-association at their exposed hydrophobic regions leading to precipitation.


Figure 25.7 A typical profile of protein solubility in solution as a function of solution pH and salt concentration. IEP, isoelectric point.


Figure 25.8 Phase behavior of proteins in solution formulation. Typical phases of physical instability of protein in solution with the addition of a precipitating agent (such as salt) or change of a precipitation inducing phenomenon (such as temperature).

The phase behavior of protein solutions, that is, whether protein solu-tion is a single-phase solution or has protein separation (two phases—solid and liquid), is affected by pH, ionic strength, and temperature (Figure 25.8). Generally, protein solubility decreases with increasing ionic strength of salts, such as NaCl and KCl (Figure 25.7). This phenomenon is called the salting out effect. This phenomenon is used to concentrate dilute solutions of proteins and to separate a mixture of proteins (if one of the proteins salts out at a lower salt concentration than the other). The added salt can then be removed by dialysis.

Organic solvents tend to decrease the solubility of proteins by lower-ing the dielectric constant of the solution (Figure 25.8). The presence of other highly water-soluble polymers in the solution (cosolutes) also tends to reduce protein solubility by their interactions with solvent molecules, thus tying up the solvent and reducing protein–solvent interactions. This phenomenon is known as the volume exclusion effect.

Hydrophobicity

Different amino acids have different degrees of hydrophobicity (Figure 25.4). Overall hydrophobicity or hydrophilicity of a protein is determined by the nature of functional groups exposed on the surface of the protein. These are the groups that contribute to protein–solvent and protein–protein interactions.


Figure 25.4 Relative hydrophobicity of different amino acids estimated based on either their side-chain sequence (scales 1 and 2) or their typical location in a globular protein structure (scales 3 and 4).

In an aqueous solution, hydrophobic regions of a polypeptide tend to point away from the hydrophilic aqueous environment to achieve the ther-modynamically least energy state of greatest stability. In doing so, the hydrophobic surfaces of a protein tend to cluster together on the inside of the protein and form multiple weak van der Waals interactions. These multiple simultaneous weak hydrophobic interactions are the single most important stabilizing influence of protein native structure, which also pro-vide flexibility of protein conformation depending on its solution environ-ment. Thus, in addition to the stabilizing interactions with the solvent on the surface, including electrostatic, van der Waals, hydrogen bonds, and ionic interactions, hydrophobic interactions within and among a protein’s polypeptide chains stabilize native protein native structure. For example, if alternating hydrophilic and hydrophobic amino acid sequences in synthetic peptides are at the optimum distances in space, the molecules coil with the hydrophobic amino acids on the inside of each coil and the hydrophilic ones to the outside. Thus, secondary, tertiary, and quaternary structures of polypeptide chains are important in determining the net hydrophobic or hydrophilic nature of the protein.

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