[26] Purification of metallothionein by fast protein liquid chromatography

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[26]

mium saturation assay coupled with anion-exchange HPLC separation of MT isoforms has proven to be a viable means of quantification of individual MT isoforms in the rat. 15 A similar combination with RP-HPLC has yet to be explored. Concluding R e m a r k s Reversed-phase HPLC is clearly a technique that has a great deal of promise for studying aspects of MT, such as its synthesis, degradation, metal composition, structure, and function. It is a rapid, highly reproducible, and sensitive technique well suited to the purification, characterization, and quantification of MT isoforms and their associated metals. The amount of sample required is considerably less than that required for classical chromatographic techniques used to isolate MT. With appropriate improvements it may be possible to further extend the utility of the analytical RP-HPLC technique to cover a range of applications from the purification of MT isoforms on a preparative scale to the isolation and quantification of MT isoforms present at low levels in tissues and physiological fluids. Acknowledgment Mention of a tradename,proprietaryproduct,or specificequipmentdoesnot constitutea guaranteeor warrantyby the U.S. Departmentof Agricultureand doesnot implyits approval to the exclusionof other suitableproducts.

[26] Purification of Metallothionein by Fast Protein Liquid Chromatography By PER-ERIC OLSSON Introduction Isolation of metallothionein (MT) and its isoforms usually involves a three-step chromatographic procedure involving a size-exclusion step, an ion-exchange step, and a buffer-desalting step using a volatile buffer prior to lyophilization and amino acid analysis.~ Characteristics used to isolate MTs by this procedure include a molecular weight of approximately l P.-E. O l s s o n a n d C. H a u x ,

Inorg. Chim. Acta, Bioinorg. Chem.

METHODS IN ENZYMOLOGY, VOL. 205

107, 67 (1985).

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

[26]

PURIFICAa'IONOF MT By FPLC

239

60002,3 and an acid isoelectric point resulting in binding to anion-exchange resins. 4 Metallothioneins are thus routinely isolated from Sephadex G-75 gel-permeation fractions containing protein with a molecular weight of approximately 10,000. Pooled fractions are further separated using anionexchange chromatography from which different MT isoforms can be eluted with a salt gradient. 5 Finally the separated isoforms of MT are subjected to a desalting step on Sephadex G-50 equilibrated with a volatile mobile phase such as an ammonium buffer to facilitate lyophilization prior to amino acid analysis. Conventional procedures for the isolation of MTs, using the above methodologies, have in some instances failed to separate i s o - M T s 6'7 or have been so time consuming as to be unsuitable for analytical purposes. Two alternative chromatographic systems have been developed to reduce isolation time and enhance the resolution of different iso-MTs. 8,9 One system utilizes reversed-phase columns coupled to high-performance liquid chromatography (HPLC) s,~° and the other system involves ion-exchange chromatography using fast-protein liquid chromatography (FPLC)9 or HPLC. 8 Both systems have been used to study the biochemical properties of MTs, achieve enhanced separation, and for antibody production? t- 16 In the present chapter the separation of teleost MT by FPLC using the Mono Q anion-exchange media is described and compared to conventional anionexchange chromatography using a DEAE-Sephadex A-25 resin.

2 j. H. R. K'figi, S. R. Himmelhoch, P. D. Whanger, J. L. Betune, and B. L. Vallee, J. Biol. Chem. 249, 3537 (1974). 3 W. F. Furey, A. H. Robbins, L. L. Clancy, D. R. Winge, B. C. Wang, and C. D. Stout, Science 231, 704 (1986). 4 M. Webb, ed., in "The Chemistry, Biochemistry and Biology of Cadmium." p. 195. Elsevier/North-Holland, 1979. 5 F. E. Reginer, this series, Vol. 104, p, 170. 6 P.-E. Olsson and C. Haux, Aquat. Toxico1. 9, 231 (1986). 7 j. Overnell and T. L. Coombs, Biochem. J. 183, 277 (1979). s K. T. Suzuki, H. Sunaga, Y. Aoki, and M. Yamamura, J. Chromatogr. 281, 159 (1983). 9 P.-E. Olsson and C. Hogstrand, J. Chromatogr. 402, 293 (1987). ~oR. W. Olafson, Int..L Pept. Protein Res. 24, 303 (1984). ~ H. Neuberger and M. L. Moniek, in "Trace Element Analytical Chemistry in Medicine and Biology Volume 5" (P. Br~tter and P. Schramel, eds.), p. 174. de Gruyer, Berlin and New York, 1988. ~2M. W. Brown, D. Shurben, J. F. de L. G. Solbe, A. Cryer, and J, Kay, Comp. Biochem. Physiol. C: Comp. Pharmacol. Toxicol. 87C, 65 (1987). ~3S. Klauser, J. H. R. K~gi, and K. J. Wilson, Biochem. J. 209, 71 (1983). ~4C. G. Norey, W. E. Lees, B. M. Darke, J. M. Stark, T. S. Baker, A. Cryer, and J. Kay, Comp. Biochem. Physiol. B: Comp. Biochem. 95B, 597 (1990). J5 K. T. Suzuki, Anal Biochem. 102, 31 (1980). ~6R. W. Olafson, W. D. McCubbin, and C. M. Kay, Biochem. J. 251, 691 (1988).

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Rationale for Column Selection The most important question confronting the investigator contemplating chromatography is that of the nature of the column to be used. By including the FPLC system the choice is broadened beyond conventional Sephadex and DEAE columns to include the Superose gel-exclusion columns as well as Mono Q anion-exchange columns. Since Sephadex G-75 has a working range of about 1 × 103 to 5 × 104 Da and Superose 12 has a working range from 1 × 103 to 3 × 105 Da, chromatography using Sephadex usually gives better separation of MTs from both high-molecularweight (HMW) material and low-molecular-weight (LMW) material subjected to matrix inclusion. On the other hand, due to the even particle size (30 pm) of the Superose it achieves shorter elution times than the Sephadex G-75 fine media (particle size 20-50/zm). Protein separation by ion-exchange chromatography depends on the differential charge between different proteins at a given pH. Although the total charges of the molecules to be separated are important, ion-exchange surfaces can readily recognize the differences between two proteins that are identical in every respect except charge distribution) Protein retention on an ion-exchange column is the result of electrostatic interactions in which the retention increases in proportion to the charge density on both the ion-exchange matrix and the protein. In contrast to a weak ion-exchange matrix (such as DEAE) a strong ion-exchange matrix (such as Mono Q) remains fully ionized over the entire pH range from 2 to 12. The strong ion-exchange resins are also thought to exhibit slightly better selectivity than the weak ones2 The Mono Q resin is based on Monobeads that bind negatively charged components through quaternary amine groups. 17 The very narrow particle size distribution of the Monobeads results in low back pressure, allowing short elution times. Materials

Chemicals. The Sephadex G-75, DEAE-Sephadex A-25, Superose 12, and Mono Q columns were obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). YM2 membranes were obtained from Amicon (Danvers, MA). Tris was obtained from Merck (Darmstadt, Germany) and ammonium bicarbonate from Sigma (St. Louis, MO). All other chemicals were analytical grade. Apparatus. Anion-exchange chromatography was carried out on an FPLC system equipped with two high-precision P-500 pumps, a liquid ~7"FPLC Ion Exchange and Chromatofocusing Principles and Methods." Pharmacia Fine Chemicals AB, Uppsala, Sweden,

[26]

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241

chromatography controller LCC-500 PLUS, a dual-path monitor UV-2, a two-channel recorder REC-482, a valve V-7, and a fraction collector FRAC-100. The Mono Q column was an HR 5/5 (5 × 50 mm) and the dimensions of the DEAE-Sephadex column were 20 X 2.6 cm. Procedures and Discussion

Sample Preparation. Metallothionein is prepared by intraperitoneal injections of juvenile perch (Perca fluviatilis) with cadmium chloride, repeatedly over a 2-week period, to yield a total dose of 3 mg Cd/kg body weight. One week after the last injection the fish are killed and the livers excised and washed in 10 m M Tris-HC1 buffer (pH 7.6). The livers are homogenized in the same buffer (20%, w/v), using a glass-Teflon homogenizer. A glass-Teflon homogenizer is preferred to a Polytron homogenizer since it results in reduced air oxidation of the proteins. The homogenates are centrifuged at 10,000 g for 20 min at 4 ° and the supernatant is recentrifuged at 105,000 g for 120 min at 4 °. The supernatant is loaded onto a Sephadex G-75 column (60 X 5.0 cm) and eluted ¢¢ith 10 m M Tris-HC1 (pH 7.6). Absorbance is measured at 254 and 280 nm and fractions of 10 ml are collected. The 254-nm peak eluting at an approximate molecular weight of 10,000 is pooled and used for anionexchange experiments using either a DEAE-Sephadex A-25 column (Fig. 1) or a Mono Q column (Fig. 2). Because MT has been found to undergo chemical alteration on repeated freeze-thawing and will be proteolytically degraded if left at 4 °, it is recommended that fresh material be used for all analytical purposes, or that aliquots be stored at - 2 0 °. Choice of Buffer. It is vital that all buffers used in the present procedure are passed through a 0.22-/~m sterile filter and are degassed before use. The Sephadex G-75-isolated MT samples in the present experiment are passed through a 0.22-pm sterile filter before being applied to the anion-exchange column. The buffers are supplemented with 0.01% NaN 3 except in the case of the ammonium bicarbonate buffer used in the Sephadex G-50 column to elute protein for amino acid analysis. The most widely used technique for eluting ion-exchange columns is by gradient at a fixed pH. Although pH gradients have been successful, they are more difficult to reproduce and generally give poorer resolution than salt gradients as both ionic strength and pH are varied. Two different gradient systems have been employed in this laboratory, one using NaC1 to generate a gradient and keeping the Tris-HC1 buffer concentration at a fixed level (data not shown), the other increasing the concentration of the Tris buffer to elute the different MT isoforms (Fig. 2). 9 Chromatography. The Mono Q column is stored in 24O/o(v/v) ethanol

242

[26]

ISOLATION AND PURIFICATION OF METALLOTHIONEINS 08

200

MT

ii

06.

G :I:

i

hi

I00

04-

LL 0 Z __0 FFZ W Z 0

I.-E

0.2

....

0 0

_.:.-..

I00

.........

I0

_ ........

300

560

ELUTION VOLUME (rnl) FIG. 1. Separation of MTs by DEAE-Sephadex A-25 chromatography was performed using a linear Tris-HCl gradient from l0 m M Tris-HC1 ( p H 8.1) to 200 m M T r i s - H G (pH 8.1). The absorbance at 254 and 280 nm was measured and 5-ml fractions collected. C a d m i u m (--), copper ( . . . . . ), a n d zinc ( . . . . . ) were measured in each fraction. The column was washed with 100 ml starting buffer after application of the sample and prior to starting the gradient. [Reprinted, with permission, from P.-E. Olsson and C. Haux, Aquat. Toxicol. 9, 231, (1986).]

and must be equilibrated before use. Equilibration is done by pumping 5 ml of 100% starting buffer (buffer A) through the column followed by 10 ml of 100% elution buffer (buffer B), and finally reequilibration with 5 ml 100% starting buffer. Chromatography is performed under the following conditions: Flow rate, 1.0 ml/min; buffer A, 10 mM Tris-HC1 (pH 7.6); buffer B, 400 mM Tris-HC1 (pH 7.6). The LCC-500 controller unit is programmed to give a stepwise Tris-HC1 gradient (see Fig. 2). Five milliliters (approximately 4 mg of MT) of Sephadex G-75-purified perch MT is loaded at time zero and the absorbance monitored at 254 and 280 nm. One-milliliter fractions are collected and the cadmium content of each fraction is determined using air-acetylene flame atomic absorption spectrophotometry (Fig. 2). In most species at least two isoforms of MT have been identified) ,4 However, when conventional anion-exchange chromatography is utilized to separate perch MT, only one MT-containing peak is observed eluting from the DEAE-Sephadex A-25 (Fig. l). Chromatography of an aliquot of

[26]

PURIFICATIONOF MT BY FPLC

243

0

O010

U

so o2

o

o ~ 0

*:''Tx-~-' I0

20

Lo 30

FRACTIONS

Fro. 2. Elution profile from Mono Q column chromatography of Sephadex G-75-purified perch MT. The absorbance of 254 nm ( - - ) and 280 nm ( - - - ) and the cadmium content ( . . . . . ) are indicated. The LCC-500 controller unit was programmed to give the following gradient after application of the sample: 0% B for 7 min, 0 to 2% B in 1 min, 2% B for 3 min, 2 to 4% B in 1 min, 4% B for 4 min, 4 to 5.5% B in 1 min, 5.5% B for 6 min, 5.5 to 100% B in 3 min, 100% B for 2 min, 0% B for 7 min. The solid line denotes the theoretical gradient. [Reprinted, with permission, from P.-E. Olsson and C. Hogstrand, J. Chromatogr. 402, 293, (1987).]

the same sample, using a Mono Q column coupled to an FPLC, results in the appearance of two distinct MT peaks (Fig. 2). The major cadmium-containing peaks from the Mono Q column are concentrated by ultrafiltration over a YM2 membrane and applied to a Superose 12 column equilibrated with 10 m M ammonium bicarbonate (pH 8.1). After final purification the proteins are lyophilized and characterized as MTs by amino acid analysis. 9 There are certain variables that will affect the elution pattern of MT. Changing the pH of the Tris-HC1 buffer results in a shift in the elution position of both MT-I and MT-2. Both isoforms elute at a higher ion concentration at a higher pH. Thus, no change in the relative elution positions is observed when isolating perch MT at different pH. However, by altering the shape of the gradient it is possible to change the relative positioning of the two MT isoforms. The first isoform elutes at about 26 m M Tris-HC1 (pH 7.6), while the second elutes at about 32 m M TrisHC1 (pH 7.6). A linear salt gradient from 10 to 400 m M Tris-HCl results in poor resolution of the two isoproteins. However, by utilizing a step gradient and flattening the ramp as soon as the first protein elutes it is possible to achieve total separation of the two proteins.

244

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[27]

Loading Capacity. The loading capacity of the Mono Q H R 5/5 column is approximately 25 mg protein. 17 However, when loading samples close to the loading capacity of the column proteins often begin breaking through with the nonretained material. On the other hand, when loading small amounts of sample recovery may decline because the surface areato-solute mass ratio in the column becomes very large, and the column is underloaded. In an underloaded column the small number of imperfections in a support that irreversibly adsorbs or denatures protein can dominate the separation? Column Maintenance. The Mono Q column should be stored in 24% ethanol when it is not used. When there is a pressure buildup in the column it is advisable to clean the column by washing with 1 - 4 ml of 70% acetic acid followed by rinsing with 10 ml doubly distilled water. If further washing is required, the column is washed with 1 - 5 ml of 2 M NaOH followed by 1 M HC1 and 2 M NaC1 with a 10-ml doubly distilled water rinse between each step. Following this the column can be reequilibrated with starting buffer or stored in 24% ethanol. Comments. An FPLC chromatographic method using the Mono Q H R 5/5 column has been developed that is suitable for analytical purposes as well as preparative isolation of MTs. 9 The Mono Q column has been used to isolate MT for antibody production. 14 Furthermore, it can readily achieve separation o f M T isoforms that will elute in the same position after DEAE-Sephadex A-25 chromatography. Acknowledgments I wish to thank Dr. R. W. Olafson (Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada) for a critical review of the manuscript.

[27] Purification of Human

Isometallothioneins

By PETER E. H U N Z I K E R

Introduction As all mammalian metallothioneins (MTs), human MT can be isolated as two charge-separable isoforms designated as MT-1 and MT-2. The purification protocol as described by BOhler and Kfigi contains two precipitation steps followed by gel-filtration and ion-exchange chromatography.l 1 R. H. O. BOhler and J. H. R. Khgi, FEBSLett. 39, 229 (1974).

METHODS IN ENZYMOLOGY, VOL. 205

Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.