BCH 612. Proteins and Enzymes. Spring 2003 T. C. Vanaman
Protein detection, assay, purification and characterization. II. Pg. 4
General Reference - Methods Enzymol. 182 "Guide to Protein Purification" (M. Deutsch, ed.). 1991.
x
x
x
x
x
c). Methods for elution.
Elution of proteins bound through charge interactions can be accomplished by changing pH and/or
increasing ionic strength.
Changes in pH or ionic strength can be done either stepwise or by continuous gradient elution.
Gradients can either be simple or complex.
The most reproducible separations are achieved with simple linear salt gradients.
More porous supports with lower cross-linking will shrink as ionic strength increases.
3). Molecular sieve or gel permeation chromatography (Stellwagen. 1990. Methods Enz. 182:
317-328.).
a). General Principle - Vt, Vo, Vi, Ve and distribution
coefficients.
x The same types of supports described above can be synthesized to have varying pore
sizes as shown in the attached table. The density of the polymer and its degree of
cross-linking dictate the pore size. Sizing resins are available for separation of
molecules as small as mono- di- and trisaccharides (100-500 daltons) up to molecules
with molecular weights in the megadalton range.
x The general principle is the same in all cases and is illustrated below.
The elution behavior of
any molecule can be
described by the equation
Partition coefficient, Kav = Ve - Vo
Vt – Vo
Ve = the volume required to elute a component, Vt = the total liquid volume of the column and Vo
= the volume of liquid outside the beads. For an example, see the attached elution profile for a Superdex
column.
Kav is strictly determined by the Stoke's radius (rotational volume) of the molecule, which is
proportional to its molecular weight and shape.
BCH 612. Proteins and Enzymes. Spring 2003 T. C. Vanaman
Protein detection, assay, purification and characterization. II. Pg. 5
General Reference - Methods Enzymol. 182 "Guide to Protein Purification" (M. Deutsch, ed.). 1991.
b). Types of gel permeation media available - selecting size separation range.
x
x
x
x
The attached Table gives fractionation ranges available for the family of carbohydrate
based supports available from Pharmacia (Sephadexes and agaroses).
Similar ranges are available in polyacrylamide based supports. However, these resins
are more hydrophobic and tend to bind proteins altering the apparent Kav vs MW
relationship.
For highest resolution, select a resin on which the molecule of interest has a Kav = 0.5.
Sizing separations are also used to exchange or remove buffer constituents in samples
of biomolecules. For "desalting" separations, it is best to chose a support where
biomolecule is essentially excluded and the 'salt' has a Kav ~ 1.
c). Sample dilution during separation.
x
x
Since sizing chromatography must not involve adsorption/desorption, the method
necessarily leads to a dilution of the input sample rather than concentration.
Dilution is usually a minimum of 3 fold.
d). Practical considerations of column geometry and operation.
x
x
x
x
x
x
Best resolution is obtained with a bed for which the height is at least 10 x the diameter.
The volume of the ample applied should also be less than 5 % of the included volume
for maximum resolution.
The lower percentage gels used for high molecular weight separations tend to compact
under pressure and must be operated at relatively low flow rates.
When necessary, columns can be operated by up-ward flow to alleviate compaction
problems.
The size of the column must be matched to the amount of material being separated.
Porous glass bead resins can be used for HPLC sizing separations on small to
intermediate scales.
Biochemistry I Fall Term, 2002
Gel Filtration and SDS PAGE Graphs
The two figures show calculated values for molecular seiving measurements.
Fig. 1 Relative elution volume is plotted vs Mr (logarithmic scale) on a gel filtration column.
The quaternary structure of some proteins we have studied in Biochemistry I:
Protein
Native Mr (Da)
Myoglobin
17,200
TIM (triosephosphate isomerase)
53,300
Hemoglobin
62,000
IgG (immunoglobulin G)
140,000
ATCase (aspartate transcarbamoylase)
307,900
#Subunits
1
2
4
4
12