Biochemistry 601

Protein Solubility, Purification, and Analysis

Reading assignments:

• Stryer, Ch. 3.

Bibliography:

• Voet, D. and Voet, J.G. (1990). Biochemistry, John Wiley and Sons, New York.

Objectives

• The ionic strength dependence of protein solubility depends on the nature of the protein and on both the concentration and nature of the salt. Differential solubility in ammonium sulfate solutions forms the basis of an effective protein purification step.
• The addition of salt can increase protein solubility (salting in) or decrease solubility (salting out).
• For a given ionic species the effectiveness of salting out parallels the effectiveness of protein structure stabilization. The Hoffmeister series rank ions on this basis.
• A protein is minimally soluble near its isoelectric point (pI).
• The rate of protein migration in non-denaturing electrophoresis depends on the mass, shape and charge of the protein.
• In SDS-PAGE, SDS and reducing agents equalize the charge/mass ratio of proteins so that they are separated only on the basis of size (by gel filtration).
• Ion exchange chromatography separates proteins on the basis of electrostatic interactions that depend on the protein's charge density, the concentration of ionic species in solution, and the pH.
• Gel filtration chromatography is both a purification technique and analytical tool that separates macromolecules on the basis of size and shape.

Protein Solubility

Salt Effects

where c(i) and z(i) are the concentration and charge of ionic species i.

Salting in

Solubility at low ionic strength increases with salt concentration. Pairing of salt ions with charged groups of the protein shields intramolecular repulsion. Precipitates tend to be irreversible, suggesting that the protein is denatured.

Salting out

Solubility at high ionic strength decreases with salt concentration. Solvent activity toward solubilization of hydrophobic solutes is reduced. When used for the purpose of salting out, solution additives are called precipitants.

Ammonium Sulfate Fractionation

At a given ammonium sulfate concentration, one protein can be very soluble while another is essentially insoluble.

Hoffmeister Series

Salt ions pair with ionized groups on proteins with varying effects on protein stability and solubilization. The most effective protein precipitants also are the most effective stabilizers. Thus, proteins may be stored in a highly stable form as the ammonium sulfate precipitate. Many enzymes are packaged and sold in this state. Good precipitants reduce the activity of the solution toward solubilization of polypeptides, exactly opposite the behavior of chaotropes.

Effect of pH

Polyacrylamide Gel Electrophoresis

Owing to their uniform shape and charge/mass ratio, DNA molecules are easily separated on the basis of size by gel filtration (or gel permeation), and their rate of migration is inversely related to the log of the molecular weight. RNA molecules typically have more variable shapes due to variable secondary structure, but these structures can be disrupted by chaotropic agents such as strong base. Proteins are neither uniform in shape nor uniform in charge/mass ratio. While non-denaturing PAGE effectively resolves protein mixtures, the relationship of size to rate of migration is not the same for different proteins. One can estimate the size of the protein by running unknown and control samples on a series of gels prepared with increasing polyacrylamide concentrations and then plotting the change in migration of the unknown and controls against polyacrylamide concentration. This method eliminates the influence of charge/mass ratio. However, rigorous analysis of protein native molecular weight is more commonly carried out by gel filtration chromatography or analytical ultracentrifugation.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a versatile and powerful tool for analyzing the polypeptide composition of protein samples, and it can provide an accurate estimate of the subunit molecular weight. SDS molecules form micelles, and each polypeptide is incorporated into a micelle by interacting hydrophobically with the detergent's alkyl tails. Variability in polypeptide shape and charge/mass ratio are eliminated by the uniform shape of the micelles and by the essentially constant ratio of 3 SDS molecules per amino acid residue. The large number of sulfate ions in the micelle overwhelms the effect of charges on the polypeptide itself.

Reagents for Protein Analysis

N,N'-methylenebisacrylamide

polyacrylamide crosslinker; Stryer, p. 46

N,N,N',N'-tetramethylethylenediamine (TEMED)

radical stabilizer

sodium dodecyl sulfate (SDS)

formation of detergent/protein micelles; Stryer, p. 47

dithiothreitol (DTT), dithioerythritol (DTE), 2-mecaptoethanol

reduction of disulfide bonds; Stryer, p. 57

phenyl isothiocyanate (PITC)

Edman degradation; Stryer, p. 55

cyanogen bromide (CNBr)

cleavage of methionine amino acyl peptide bonds; Stryer, p. 56

trypsin

cleavage of lysine and arginine amino acyl peptide bonds; Stryer p. 56

iodoacetate

modification of cysteine (also reactive to Met, His, Asp, Glu, Lys); Stryer, p. 57

performic acid

modification of cystine (also reactive to Cys, Met); Stryer, p. 58