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115:412/508 Proteins and Enzymes
spring 2002 Hydrophobic chromatography, metal ion chromatography, gel filtration, hplc Hydrophobic
chromatography Just as proteins can stick to an ionic adsorbent by charge interaction, they can stick to a hydrophobic material by hydrophobic interaction with nonpolar regions of their surfaces. This was discovered during early work on affinity chromatography, when control adsorbents with hydrophobic spacer arms but no specific ligand at their end turned out to adsorb proteins too. The hydrophobic groups used include alkyl (octyl-Sepharose), phenyl (phenyl-Sepharose) and alkylamino chains. The capacity is high, and adsorption is strongest at high salt concentration, so a sample may be applied immediately on redissolution after (NH4)2SO4 precipitation, though in that case the exact salt concentration might be difficult to repeat. Proteins are eluted by decreasing the salt concentration, or in extreme cases by adding ethylene glycol or even ethanol to a very low ionic strength buffer. Resolution is not as good as in ion exchange chromatography, because a changes slowly, and the mechanism is similar to (NH4)2SO4 precipitation, so it may not purify much more if that method has been used carefully. It is best used fairly early in a purification procedure. A variant of hydrophobic chromatography is "T-gel", agarose which has been treated successively with divinylsulfone and b-mercaptoethanol, producing a -CH2CH2SO2CH2CH2SCH2CH2SH side chain which for unknown reasons has high and fairly specific affinity for immunoglobulins. Agarose
and even cellulose act as hydrophobic adsorption materials at high enough
salt concentration, a little below the (NH4)2SO4
concentration required to precipitate the protein, which may be applied
in this concentration of (NH4)2SO4,
or precipitated in presence of agarose, onto the agarose rather than
as a precipitate, and poured onto the top of an agarose column and eluted
with a decreasing salt gradient. Metal-binding chromatography This can be a general approach for proteins with exposed histidines, cysteines or carboxyl groups near each other, but in practice it is mainly for cloned proteins. If you are working with a cloned protein, you can add to the cloned gene a short sequence which facilitates purification. The method which is becoming more and more used is to add a sequence of six or so histidines at the C-terminus. These bind well to divalent cations of transition metals such as nickel. A column is prepared by attaching nitrilotriacetic acid to a solid support. This binds nickel ions tightly; the resin is washed with 5 mm imidazole to remove unbound nickel. The fusion protein, perhaps denatured in 6m urea to ensure that the hexa-His sequence is exposed, is applied to the column. The column is washed with dilute imidazole, then more concentrated imidazole to elute the desired protein. An example of this is in a paper by Witzgall et al., Anal. Biochem. 223:291-298 (1994), who in addition attached at the N-terminal end of the protein a DNA-binding domain from the yeast transcription factor GAL4 and an eleven amino acid peptide from bacteriophage T7 to which there are commercially available antibodies; this gave them a way to visualize the fusion protein on western blots. Other examples of genetically modifying the cloned protein for purification are using a (his-asp)n sequence to bind to nickel, or a poly-arginine sequence to make the protein cationic. Added C-terminal sequences can often be removed by an appropriate carboxypeptidase - A for histidines, B for arginines - if this does not chew on into the native protein. Some
variations are: mercuric ions bound tightly to immobilized sulfhydryl
groups, which can bind proteins by their exposed sulfhydryl groups -
elution is with excess free SH compound such as mercaptoethanol; and
Fe+++ bound to iminodiacetic acid, which binds phosphoproteins
by the phosphate groups. Other general
adsorbents These include mixed-function groups attached to agarose, such as sulfanilic acid and arginine, which have shown good capacity and some selectivity; phenylboronate-agarose, whose -B(OH)3- group forms reversible covalent bonds with vicinal diols, and thus selectively adsorbs glycoproteins, or other proteins which bind tightly a ligand with a carbohydrate moiety such as NAD+ or AMP. Column dimensions The manufacturer generally specifies the binding capacity of the material, though after it is swelled up and separated from its original bottle you will lose track of this. Large proteins cannot penetrate as many pores as small ones, so the capacity per unit volume or per weight of adsorbent will be considerably less. For a simple stepwise elution procedure, especially concentrating the protein from a dilute solution, one can adsorb on as much as half the column, since little further fractionation is expected; for careful separations with gradients the total column volume should be 5 to 20x the volume it takes to adsorb the protein. In a perfect world the shape of the column wouldn't make any difference to the elution, which in theory depends only on volumes of column material and buffers. But the world isn't that perfect; a given distance of diffusion or irregularity of elution makes much more difference to a short fat column than to a long thin column (see handout). But a short fat column runs much faster. Thus again for simple stepwise elution, including concentration, a short fat column is used, sometimes as wide as high; for careful separations with an extended gradient a long thin column is used, though this is not so critical as in gel filtration. Ultrafiltration This is the filtration of a protein solution
through a membrane with pores small enough to retain the protein of
interest. It is a two-phase
method - what is retained and what passes through the filter - but I
mention it here in preparation for gel filtration.
To happen in a reasonable time it requires either gas pressure
on the solution above the filter (for large volumes) or increase of
gravitational force by centrifugation (for small volumes).
It is usually used just to concentrate a dilute protein solution,
such as a crude culture broth, and sometimes for dialysis by diluting
the concentrated solution with dilute buffer and concentrating again
until practically all small molecules have been flushed through the
membrane. It is sometimes used as a purification method, with membranes which
retain the protein of interest but pass through smaller ones; this
is particularly appropriate if the protein is very large, both because
greater purification is achieved and because the filtration will be
faster through the larger pores involved.
The biggest problem is clogging of the pores by protein accumulating
on the membrane surface; Amicon makes apparatuses which provide for
continuous stirring while the protein is filtered under pressure.
Use of this as a purification method is most appropriate when
you want to concentrate the solution anyway. Gel filtration I have mentioned this procedure many times, you probably
have at least some knowledge of it, and you would surely have used it
by this time in a purification; so I shall not spend long on it. The principle by now seems simple. The gel filtration material (Scopes complains
that it doesn't have to be a gel and is not really filtration, but it's
too late to attach a different name) is porous, with pores the size
of protein molecules. Large molecules, too large to enter any of
the pores, pass down the column in the space between the gel particles,
V0 = 0.3 - 0.38 of the total volume of the column, and therefore
are eluted first, when that volume has passed through;
very small molecules enter all the pores, and therefore spend much of
their time not moving and elute only when a volume Vt virtually
the entire volume occupied by the bed has passed through; intermediate
size molecules enter some of the pores, and are eluted somewhere in
between. I sometimes compare the situation to search
of a neighborhood by policemen in a squad car, rapid but of course
incomplete, to a search entering every room, which would take a lot
longer. Here we want to separate fat policemen from
thin policemen according to what narrow doors they can pass through. Gel filtration of course is also used to separate
proteins from salts such as ammonium sulfate, using a small-pored gel
such as Sephadex G-25 or BioGel P10 which excludes all proteins; it
is much faster than dialysis, and whether it dilutes more or less depends
on how much expansion you allow in dialysis; but it is less applicable
to really large volumes, and requires a little more attention. The first gel filtration materials were the Sephadexes, cross-linked dextrans sold by Pharmacia; the larger pore sizes are very easily crushed by gravity and flow and little used nowadays, because of the availability of stronger materials which do not crush easily. See Table 6.1 from Scopes (handout). Note that having a wide fractionation range, as with many of the more modern materials, is not necessarily an advantage; the fractionation occurs within 55% of the column volume, and if a wider range of molecular weights are fractionated, any given pair, say proteins of mol. wt. 100,000 and 150,000, will be closer together on a material of wider fractionation range. You might use a wide fractionation range material to give an initial cut, eliminating much larger and much smaller proteins, and then use a narrower range material for best separation of these two. The best separation occurs for molecules eluting at about 0.6 column volume, but the peaks get broader the later they come off, and if separation from a slightly smaller protein is most wanted, you may want to use a smaller-pored column from which your protein elutes earlier (Fig. 6.8). There are no peak sharpening mechanisms on a gel filtration
column, so for maximum resolution you want to apply your sample in as
small a volume as possible, short of having viscosity effects - generally
at a protein concentration between 10 and 30 mg/ml. Scopes suggests determining the column size as follows: if m is
the amount of protein in mg, the column diameter is given by the cube
root of m/10 cm, and the length
should be 30x the diameter. A
heavy solution, such as an ammonium sulfate precipitate, may run ahead
between the beads rather than equilibrating with them, leading to a
diffuse leading edge. Scopes suggests flowing the sample upward from
the bottom, which can be done either using a pump or having the effluent
line below the lowest point of the application line, to avoid this;
but in my experience materials such as Sephadex G-75 may shrink in the
high salt concentration, leaving a pool of sample at the bottom of the
column which doesn't run in. So
I would do just the reverse of what he suggests: I would apply the sample
to the top of the column in a 'right side up' position, let it run in
slowly, then after it is all in the gel invert the column and elute
it upwards. Running a column from the bottom upwards is good for the higher
Sephadexes and other gels subject to crushing, because the flow tends
to oppose gravity rather than working with it in crushing the gel. But with the modern cross-linked agaroses
and dextrans this is hardly a problem anyway.
Note also that resolution can be lost after the column, if there is too large a space below the bed where
the solution is funneled into tubing, or if the tubing going to the
fraction collector is too long or wide; you want to use the smallest
tubing that does not itself become a limitation on the flow rate of
the column. In principle separation depends only on the volume of gel the sample passes through, so that a short fat column could be used as well as a long thin column; but in practice the effects of uneven flow, as well as diffusive broadening, are much worse in a short fat column, as shown in Fig. 6.9. The column material is supposed to be inert toward the
protein, but there are various exceptions.
Most column materials have a few free carboxyl groups, due to
oxidation of carbohydrate or hydrolysis of acrylamides; chromatography
should therefore be run in a high enough buffer concentration to prevent
ionic adsorption to the column material, generally 50 mm,
unless you are also desalting to a lower ionic strength. Polyacrylamide materials such as BioGels, and
even cellulose and agarose at very high salt concentration, can adsorb
protein by hydrophobic interactions; if this happens one should chromatograph
at lower ionic strength or on a dextran polymer. On the other hand, proteins whose natural role is to bind to polysaccharide
materials, such as cellulases and amylases, will probably bind to dextrans
and be retarded, eluting much later than expected from their molecular
weight, even after the salt fraction. Specific interactions of this sort, a sort of low-grade affinity
chromatography, can of course be taken advantage of in a purification
- elute from polyacrylamide at the protein's proper molecular weight,
then separate it from other proteins of the same mol. wt. by chromatography
on Sephadex. HPLC
of proteins HPLC now stands for "high performance liquid chromatography" though HP could also be said to stand for "high pressure". It was first introduced for rapid chromatographic separation of small molecules, using small columns of very small particles 5 or 10 µm in diameter. The smaller the particles, the greater the number of "theoretical plates", the more rapid the attainment of diffusional equilibrium between stationary and moving phases, and therefore the better the separation for a given length of column (read about this in Scopes, pp. 105-111. In previous years I have had Dr. Ward lecture on protein hplc, but he would spend two full lectures on the theory and not get to the practice.) These columns consequently yield good separation and high resolution even though run rapidly. One advantage of fast operation is that they can be run at room temperature without denaturing the protein, because the protein comes off in 5 to 60 min. However, although the linear rate of flow is high, say 20 cm/hr, because the diameter is small, say 7.5 mm, the optimal volume rate is low, say 0.15 ml/min. Consequently the columns are usually run at a volume rate much greater than the optimal for reaching diffusional equilibrium. Optimum flow rate decreases with bead size - Scopes says increases inversely, which is confusing - and pressure to achieve the same flow rate increases with 1/r2 of the bead, so that halving the bead size leads to an eight-fold increase in the pressure necessary to run the column at a given rate. Thus the columns for full hplc are constructed of stainless steel; Pharmacia has a system with plastic columns of intermediate size, called Fast Protein Liquid Chromatography, useful for slightly larger quantities. It is run at room temperature, but doesn’t take long, so denaturation in the warm isn’t usually a problem. For small organic
molecules the particles could be based on strong inorganic materials
such as silica, and organic solvents could be used; however, high pressure
was required to push solvent through columns filled with small particles. For protein chromatography it was necessary
to develop materials both strong enough to stand the pressure and porous
enough to have a high surface area for adsorption, or for gel filtration. These materials are very expensive, and the
operator becomes paranoid about not having the column clogged by precipitated
protein; the sample is usually passed through a 0.2 µm filter just before
going onto the column. The bottom line
is that hplc is a high resolution, but low capacity method. Its main uses, unless you have thousands of
dollars for preparative columns, are as a final step in purification
to remove the very last impurity, or for separation of peptides after
proteolytic digestion of the protein, when biological activity no longer
matters and organic solvents and 0.1% trifluoroacetic acid can be used,
often in reversed phase mode, more appropriate because in denatured
peptides all the hydrophobic groups are exposed.
Such separation of peptides can be used as a "fingerprint"
- how many peptides are produced by tryptic digestion, which one changes
mobility in a mutant protein - but is most important for sequencing
the protein. |