Download General Reference - Methods Enzymol. 182 "Guide to Protein

BCH 612. Proteins and Enzymes. Spring 2003 T. C. Vanaman
Protein detection, assay, purification and characterization. II. Pg. 1
General Reference - Methods Enzymol. 182 "Guide to Protein Purification" (M. Deutsch, ed.). 1991.
3. Methods for protein purification.
a. Fractionation by precipitation methods (Ref'n – S. Englard and S. Seifter. 1990. Methods Enz.
182:285-300.).
1). Properties of proteins which dictate solubility under
various conditions.
x Charge properties and isoelectric point
x Hydrophobic/hydrophilic character
x Size
x Thermal stability.
2). Isoelectric point and pH precipitation.
x Proteins have their lowest solubility and greatest
stability at the pH equal to their isoelectric point.
x The pI of a protein is determined essentially by it's
content of Asp + Glu vs His + Lys + Arg.
For a protein with 3 Asp, 3 Glu, 2 His, 4 Lys and 3 Arg, there
are 6 total – charged side chains and 9 total + charged side chains.
Thus, the pI will be the pH at which only 6 of the 9 His + Lys + Arg
residues are protonated. Given their pKas, this will be governed by the
protonation of lysyl residues (pKaHis # 6, pKaLys # 9.0, pKaArg # 13)
since the form with no net charge would have 3 of the four Lys
residues protonated. Using the Henderson-Hasselbalch equation,
= 9.0 + Log 1
= 9.0 - 0.48 = 8.52
pH = pI = pKa + Log10[conjugate base]
[conjugate acid]
3
This protein would be least soluble at pH = 8.52.
3). Salt fractionation - Salting in and salting out.
x All proteins require some counter-ions (i.e. salt) to be soluble in aqueous media.
Therefore, protein solubility increases with n salt concentration at low ionic strength.
x At higher ionic strength, protein solubility generally decreases with n salt concentration
due to reducing the activity of water and neutralization of surface charge. Each protein
has a distinct solubility profile as a function of salt concentration defined by:
Log S(mg/ml) = E - Ks(ionic strength/2) where Ks and = E are constants.
x Different types of salts effect the solubility of proteins to different extents. Most widely
used in protein fraction are sulfate salts, particularly ammonium sulfate (NH4)2SO4.
x In general, the larger the protein, the lower the salt concentration required to precipitate it.
BCH 612. Proteins and Enzymes. Spring 2003 T. C. Vanaman
Protein detection, assay, purification and characterization. II. Pg. 2
General Reference - Methods Enzymol. 182 "Guide to Protein Purification" (M. Deutsch, ed.). 1991.
4). Thermal denaturation.
x Proteins differ in their thermal stability and ability to renature after thermal
denaturation. Calmodulin is an excellent example of a protein that can be purified by
thermal denaturation.
calmodulin
% Native structure
Average
protein
0
100
Temp, qC
In general, smaller, highly charged proteins are stable to higher temperatures than large, more
hydrophobic proteins. Conditions are chosen such that the protein of interest is not denatured but other
less stable proteins are. In the case of calmodulin, crude tissue homogenates can be treated at 100q for 10
– 15 minutes and calmodulin recovered in high yield because it renatures very readily.
x A major limitation to the use of this technique is the action of proteases which are
inherently resistant to denaturation
5). Agents which alter dielectric constant - organic solvent precipitation.
x
Polar organic solvents such as aliphatic alcohols and ketones reduce protein solubility
in two ways.
1. They replace H2O interactions with the protein surface decreasing hydration
and increasing hydrophobic surface promoting aggregation.
2. They lower the dielectric constant of H2O reducing solubility.
x
Most commonly used are methanol, ethanol, acetone and poly-alcohols such as
polyethylene glycol.
Acetone, ethanol and methanol are also excellent lipid solvents and are routinely used
to de-lipidate tissue extracts (e.g., preparing acetone powders) prior to commencing
purification procedures.
Protein denaturation is the major drawback to this approach and is largely
circumvented by using polyalcohols.
x
x