Affinity Chromatography of Lactate Dehydrogenase An Experiment for the Undergraduate Biochemistry Laboratory Alexander J. Anderson Department of Medical Laboratory Science, Queensland Institute of Technology. 2 George Street, Brisbane 4000, Queensland, Australia 01buffer pH 9.0. Just before use pipet 24 mL of this solution into a vial containing 80 mg of NADt, swirl gently to dissolve, and equilibrate to the temperature being used for the enzyme assay. The protein assay reagents (Bradford5)are conveniently available as a kit from Bio-Rad Laboratories, Hornsby, NSW.
Affinity chromatography has become a very important method in modern biochemistw. capable of aivina rapid purification of proteins, enzymes-, indd'otherbiochemicals in high yield and under mild conditions (Lowe'). The techniquecan use the affinity of a protein for a biologically specific ligand, pseudo-affinity as with the binding of dehydrogenases and kinases to triazine dyes, or a chemical affinity such as the hinding of glycoproteins to horonic acid matrices. Few experiments in affinity chromatography for undereraduates in biochemistrv have been published.. ~ .ossiblv because the gel matrices are usually quite expensive. Boverz has oublished details of an exoeriment exph)itine the chemical affinity of glycosylated hemoglobin fo; a boronic acid gel. As part of a course on biochemical methodoloav for undergraduate students majoring in biochemistry;ihis experiment was designed to illustrate the technique of enzyme purification byaffinity chromatography. The work of Ramadoss et al.3 on the affinity chromatography of dehydrogenases and kinases on Cibacron Blue ggarose was used as the basis for design of this experiment.
Running the Column Onehalf milliliter of gel (parked vdume) is packed into a small disposable column, and U.5 ml. of the rat muscle extract is applied. The column is washed with 5.5 mL of wash buffer. and 2 X 3 mL fractions (fractions 1 and2) arecollected.The enzyme is then eluted with 6 mL of elution buffer, again collecting 2 X 3 mL fractions (fractions3 and 4).
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Assay of the Fractions Enzyme activity in the original homogenate and in each fractionis assayed by mixing 2.9 mL of substrate solution with 0.1 mL of enzvme solution and follawine the reaction at 340 nm (Henrv . , et n l 4 1 If the "hewed rate ir t w fast, dilurr the enryrnr, and =*say again. Rates between 0.01 and O 1 absorhnnce unitr prr minute are needed fur accuracy. Protein concentrations in the original extract and each column fraction are also measured, using the method of Bradford5with a gamma-globulin standard.
Experlmentai Details
Results and Dlscusslon Two students working as a nair can easilv comnlete the experiment in the three-%our session. The results presented in the table were actual results obtained b v a ~ a i of r students and are typical of results obtained by"all'students. The purification factor ohtained is usually between 13 and 20, while the yield of enzyme is almost always close to 100%. Variations from these figures are usually due to insufficient dilution of the enzyme solutions before assay. It is important
Preparation of Rat Muscle Homogenate Kill a rat, and collect some skeletal muscle tissue. Homogenize 1.0 g of muslce tissue with 15.0 mL of ice-cold Tris-HCI buffer, 0.05 molL pH 7.5, containing 1 rnrnol/L each NazEDTAand 2-mereaptoethanol. Centrifuge for 15 min at 25,000 gin a refrigerated highspeed centrifuge, and discard the pellet. The supernatant should be keut on ice and used the same dav. Alternativelv. ~, ,. centrifuee the 25.0Ml g supernatant at 150,OLlOr: fm fiO min in a preparatiw ultra. c~ntrifuge,and store the resulting supernatant at -20 'C or below. The muirle ertrmr will retain its activity for wveral werks nt -20 'C or several months at -70 'C Reagents Required The wash buffer is 0.05 molL phosphate pH 6.9. The elution buffer is prepared just before use by mixing 60 mL wash buffer, 4.6 mg NADH, 54 mg oxamic acid, and 0.6 mL of 1.0 m o m NaOH. This amount is sufficient for 10 exoeriments. The gel (Cihacron Blue aearose (4%crosslinked. tvoe 3000-CL).S i m a che&cal Comuanv. i i . Louis. MOI 1s quilibrked with ;he ;,ash h u f f ~prior r ti, 11; cummrncement of the class. The enzyme aarsy solution (Henry ec ai.') consists of 0.72 mol/l. I.-lactatr in 0.1 mdiL 2-amino-prupan-l-
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Lowe. C. R. An Introduction to Affinity Chromatography; NorthHolland: Amsterdam, 1979. Boyer, R. F. Modem Experimental Biochemistry; Addison-Wesley: Reading, MA, 1986. Ramadoss, C. S.; Steczko, J.; Uhlig, J. W.; Axelrod. B. Anal. Biochem. 1983, 130,481-484. Henry, R. J.; Cannon, D. C.; Winkelman. J. W.. Eds. Clinical Chemistry, Principles and Technics, 2nd ed.; Harper and Row: New York. 1974: p 824. Bradford, M. Anal. Biochem. 1976, 72,248-254.
Purltlcatlon 01 Lactate Dehydrogenase on Cibacron ~ i u Agarose e
Sample
Volume (mL)
Original extract Fraction 1 Fraction 2 Fraction 3 Fraction 4
0.5 3.0 3.0 3.0 3.0
At 25
Total Protein
Total Activity'
Specific Activity
Yleld
Purification
fW)
(1.U.)
(I.U./mg)
(%)
factor
3.69 1.16 0.111 0.194 0.065
1310 0 0 1296 0
355 0 0 5687 0
(100) 0 0 98.9 0
(1)
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18.8
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'C.
Volume 65
Number 10
October 1988
901
that the students check that their observed rates are within the allowable range given in the section ahove. The low levels of protein in the column fractions require the use of a sensitive assay procedure to measure protein concentration. The biuret assay method is not sufficiently sensitive, while the NADH present in the elution buffer gives a high background a t 280 nm. The Bradford assay has performed very satisfactorily in these laboratories, hut the Lowry assay (Boyer2) would probably give good results. The recovery of protein is well below 100%.The explanation of this phenomenon is a good topic for discussion when
902
Journal of Chemical Education
the students prepare their laboratory reports, where comparison may be drawn between the use of rather nonspecific ligands such as horonic acid and triazine dyes, and specific active-site-directed ligands. The use of bovine serum albumin as a column pre-wash (Ramadoss et aL3) can also be discussed. Finally, it should he pointed out that the source of the enzyme is not limited to a fresh rat muscle extract, or the enzyme to lactate dehydrogenase. Readers are referred to the work of Ramadoss et a1.3 as a guide t o establishing conditions for the affinity chromatography of other enzymes.
