Purification of Milk Whey dactalbumin by Immobilized Metal-Ion Affinity Chromatography Rodney F. Boyer Hope College, Holland, MI 49423 The current increase in research activity focussing on characterizing and cloning nucleic acids has not lessened the importance of prntein purification and characteri7ation. Indeed. the advent of the new molecular hiologv -. .. with its capahility to synthesize numerous recombinant proteins has made i t even more imoortant for students to understand and experience the modern techniques of protein purification. Most protocols that apply extraction techniques and various chromatographic methods to the purification of a protein are tedious and time-consuming and, therefore, not appropriate for the typical 3-h or even 6-h undergraduate laboratory period. With this in mind, we have designed a simple, rapid, and relevant biochemistry experiment that introduces students to one of the most modern and potentially most useful chromatographic methods. In this experiment, students apply the technique of immobilized metal-ion affinity chromatography (IMAC) to the isolation of a-lactalbumin from milk whey. In addition to gaining experience in column chromatoeraohv, . .. students are introduced to isoelectric precipitation, centrifugation, and spectroscopic analysis of proteins. A few affinity chromatography experiments have been published ( 1 4 ) ;however, none applies the method of IMAC. This experiment has several advantages, including the availability of a convenient and inexpensive source of the protein, fewer health hazards than those encountered with other biological fluids (serum, urine), and it does not require the sacrificing of animals.
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Background Theory
Biochemical investigations of all biological processes require, at some time, the isolation, purification, and cbaracterization of a protein. There is no single technique or sequence of techniques that can be followed to purify all proteins. The best purification procedure is one that yields a maximum amount of the purified protein in a minimum amount of time. The basic steps include (1) preparation of a crude extract by sonication, homogenization, etc.; (2) fractionation by physical methods (centrifugation); (3) fractionation by differential solubility (pH-, salt-, or heat-precipitation); (4) chromatography by ion-exchange, adsorption, gelfiltration, and affinity, and (5) isoelectric focussing. Each of these steps has been discussed in detail in the literature (57). Chromatography bas been and will continue to be one of the most effective techniques for protein purification. The more conventional procedures (ion-exchange, adsorption, gel-filtration) rely on rather nonspecific physico-chemical interactions between a stationary support and protein molecule. These techniques, which separate proteins on the basis of net charge, polarity and size, do not display high specificity. AffinityChromatography and MAC The ultimate in selectivitv is offered by affinity chromatography-the separation of proteins on the basis of specific bioloeical interactions (8).The biological function displayed by mist macromolecules (enzymes,-nucleic acids, polysaccharides, antibodies, transport proteins, receptor proteins, 430
Journal of Chemical Education
etc.) is a result of recoenition and reversible binding with specific molecules callei ligands. This type of interaction is illustrated bv. ea. 1 in which a macromolecule A forms a complex with B, a smaller molecule or ligand:
+ B =+ A:B -Biological
A
response
(1)
In a biological system, the formation of the complex often triggers a response such as catalytic breakdown of a substrate, control of a metabolic process, immunological action, hormone action, or membrane transport. The biological response depends upon proper molecular recognition and bindina as shown in eq 1.The most common example of eq 1 i.. s the interaction ~~~~-~~~~~ that occurs between an enzvme molecule and a substrate with formation of an enzyme:substrate comolex. The resultine bioloeical resoonse from this interaction Fithe tr&format';on of ;he substrate to a product. Only the first step in eq 1, reversible formation of the complex, is of concern in affinity chromatography. In oractice. affinitv chromatographs the prepara.~ . . requires . tion bran i n s v ~ u b l e ~ t a t i o n phase, a r ~ to whirh appropriate lieand molecules are covalently affixed. The affinity support i l t h e n packed into a column through which a mixture containine the desired macromolecule is allowed to percolate. Only the macromolecules that form a complex with the affinity support will be retarded in their movement through the column. (On occasion, a protein may bind to an affinity gel because the protein interacts favorably with the support matrix..not the attached lieand. The presence of nonspecific binding can be tested by using the identical affinity support wlfhout the soecific lieand attached.) After all nonbinding molecules have been washed from the column, the desired macromolecules are eluted hv disruption of the com. gentle . plex. IMAC, which was introduced by Porath in 1975, is a modified form of affinity chromatography in which the affinity ~
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Figure 1.Structure of Me-IDA agaroseaffinlty support.Me =metal ion such as Cu(1l). Zn(ll), Ni(1l) w Fe(l1);Gel = agarose. (Adapted from ref 15.)
support consists of metal-ions chelated to an insoluble support or matrix (9, 10). The chemical structure of the most common metal ion support, iminodiacetic acid (IDA) covalently linked to a polymer such as agarose, is shown in Figure 1. The metal ions (Me) which are most effective for protein separations are Cu(II), Zn(II), Ni(I1) and Fe(II1). Using this solid support, proteins are separated according to their ability to bind to the immobilized metal ions. The binding of proteins to these supports is due to the presence of electrondonatingamino acid side chains on the surface of the protein molecules. The most important electron-rich groups on the proteins are the imidazole ring from histidine, the indole ring from tryptophan and the sulfhydryl group from cysteine. These electron-donating groups are able to displace weakly bonded metal ligands such as water. As shown in Fieure 1. a tvuical metal ion chelated to the affinitv sumort wiil havk three adsorption sites where electron:don&ng aroups on the protein mav bind. A protein, thereby, has the potential to bind to the metal ion b; multipoint attkhment. This results in relatively strong coordination bonds. The bound protein may then be released from the column by eluting with a solution containing a free ligand that can displace the protein and bind to the metal ion. The most effective eluting ligands are imidazole, ammonia, pyrophosphate, and amino acids. Alternatively, proteins may be eluted by a p H gradient. ry-lactalbumin and Other Milk Proteins
Milk is com~osedof several proteins includina, the caseins, j3-lactogfobulin, a-lactalbGmin, albumin, and various immunoglobulins. The principal proteins of milk are the phosphoproteins called caseins. However, the most well studied milk protein is probably a-lactalbumin. A crude preparation ofol-lactalb&in wasfirst obtained from milk in 1899. a-Lactalbumin, which is present in concentrations averaging 1mg/mL of milk, wasat first thought to serve mainly a nutritional function. We now know that this relatively small protein (mw = 15,500; 129 amino acids) is an essential component of the lactose synthetase system (11,12). Although several methods for the isolation and purification of a-lactalbumin have been reported in the literature, most are tedious, consisting of several steps, including p H and salt precipitation, centrifugation, dialysis, gel filtration, ion-exchange chromatography and crystallization (5,13,14). IMAC now makes i t possible to purify milk whey a-lactalbumin in a few steps (Fig. 2). The caseins are precipitated a t p H 4.6 with heating followed by centrifugation. The primary proteins remaining in the whey (supernatant) are a-lactalbumin and @-lactoglobulin.After filtration, the whey is applied directly to a metal affinity column. The column support described in this experiment is Cu(I1)-IDA agarose (15). The specific amino acid side chains on a-lactalbumin that bind to the immobilized Cu(I1) are not known. However, i t should not be surprising that a-lactalbumin binds to the column since the protein under normal physiological conditions is a metalloprotein; it carries one Ca(I1) per molecule of protein. In addition, both Mn(I1) and Zn(I1) bind to the protein, but with much less affinity than Ca(I1) (12). The affinitv-bound a-lactalbumin is eluted from the column with ;buffered solution of imidazole. The imidazole, which is in a hieh concentration relative to the motein. dis~laces the electi&rich ligands of the protein from the nietai. This results in release of the protein which can then be collected and analyzed. The elution of the protein is monitored by continuous absorbance measurement of the eluant at 280 nm. Practical Aspects of the Experiment
The ideal source is raw, unprocessed bovine milk; however, the results are essentially the same if skim milk from a grocery is used. I t is more convenient if this experiment is
Skim milk
supernatmt
Lipid precipitate (discard)
1) adjvrt to pE = 4.6 2) heat at 40' 3) centrifuge
I
I I
supernatant (whey)
Precipitate of raseina [discard)
Siter a h apply
to affinity column
Figure 2. Flowchart lor isolation and purificationof
0-lactalbumin from milk.
completed with an automatic UVflow cell monitor and fraction collector. Student time is not meatlv increased if fractions are collected by hand. The prepa;ation of whey requires approximately 3 h, and the affinity chromatography requires another 3 h. If only 3-h laboratory periods are available, students can prepare whey the first period, freeze the whev. and c o m ~ l e t the e ex~erimentdurine the next oeriod. ~lternatively,;hey can bk prepared by 'the instructor or teaching assistants and students can complete the affinity chromatography in one 3-h laboratory period. Affinity gels are relatively expensive: howeverreach student reauires onlv a small amo&,8nd it can he recicled. he IDA-agarose can he purchased in bulk furm (from Sigma. Pierce or Supelco) and packed into acolumn, or 0.8- X %cmprepacked coiumni of IDA-agarose are available from Pierce (Rockford, IL).
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Materials and Supplles Skim milk, approximately 10 mL for each student; 12 M HC1; 0.5 M HCL; high-speed centrifuge; pH meter; heatedstirrer; syringe oreoaeked or oreeartridee filter. 0.45 am: IDA aearose column... . pared ~ a ~ l ~ ~ ~10.8 c oX 8-~0cml;F3ufferA: l u m ~ 211mhITr:s,~.'~ M UaCI, pH 7.0;Buffer H.20 mhl l'ris.0.5 11 NaCI, 2u m\l imidazole, pH 7.0: CuSO, in H.0, 1 M: IIV monitor and fraction collector. U V speetrophotometer. Procedure Preparation of Bovine Milk Whey
Obtain 10 mL of milk, and centrifuge for 45 min at 16,000 X g. (During centrifugation,proceed to the section on Affinity Chromatogmphy). Decant the supernetent into a beaker leaving the sedi,.mentand floatinglipid layer in the centrifuge tube. Adjust the pH of the supernatant to 4.6. This should be done in two steps: (1)adjust to pH 5 with dropwise addition of 12 M HCI, (2) then adjust to pH 4.6 with dropwise addition of 0.5 M HCI. Heat the coagulated solution, with constant stirring, at 40 'C for 30 min. Centrifuge the mixture at 16,000 X g for 30 min. Collect the supernatant (whey), and clarify by filtering through a 0.45-rrm syringe cartridge filter. The whey may be stored for shorter periods of time in crushed ice. For longer periods (days)freeze the whey. Affinity Chromatography
Obtain a prepacked column and clamp to a ring stand. If a prepacked column of IDA-agarose is not available, prepare one by clamping a 1- X 8-10-em glass column to a ring stand and pouring in about 2 mL of the IDA-agarose gel slurry. Be sure the column outlet is closed. Allow the gel to settle (should be 1-2 cm high). To protect the surface of the gel, cut a circle (from filter paper) that has a diameter slightly less than the inside diameter of the column. Put this into the column, and allow it to slowly settle onto the top of the gel. Open the outlet of the column, and allow most of the solution to Volume 68
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May 1991
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pass through. Close the outlet, and add Buffer A to fill the column. Wash the column (prepacked or handmade) with 10 mL of Buffer A. Any flow rateup to 2mL/minispermissihle.Since thiseluant will he discarded, it may he collected in a single container. Do not allow the column to go dry. To load the column with copper, drain Buffer A to a level just slightly above the gel top. Add 0.5 mLof 1 M CuSO4 solution to the column, and allow it to enter the gel. Once all the CuS04 is in the gel, immediately add Buffer A, adjust the flow rate to 2 mL/min and continue to wash the column until all excess Cu(I1) has beeneluted (about 25 mL). Theresin willchange from white toa pale blue color. Attach the column outlet to a UV flow cell, fraction collector and recorder, if available. Alternatively, collect fractions hy hand. Allow Buffer A to drain just to the top of the gel, and add 0.5 mL of whey directlv to the too of the eel. Beein to collect 1 mL fractions in individual test tulies. Let t
