burnal qf’chrotnatography, 405 (1987) 321-336 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CHROM. 19 731

FRACTIONATION OF PLANT AND ANIMAL HIGH MOBILITY GROUP CHROMOSOMAL PROTEINS BY ION-EXCHANGE AND REVERSED- PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

STEVEN SPIKER*, MARCIA BATES and LAURA J. ARWOOD

Department of Genetics, North Carolina State University, Raleigh, NC 2769S-7614 (U.S.A.)

(Received May 18th. 1987)

SUMMARY

A method for purifying wheat high mobility group (HMG) chromosomal pro- teins using a combination of weak cation-exchange and reversed-phase high-per- formance liquid chromatography (HPLC) is described. Previously reported HPLC systems devised for fractionating HMG proteins of vertebrate animals are not effec- tive for the plant proteins. The system described here can be used for the fractionation of both plant and animal HMG proteins.

INTRODUCTION

The high mobility group (HMG) chromosomal proteins were originally dis- covered as contaminants in preparations of calf thymus Hl histones1,2. The proteins can be put into two groups based on both molecular weight and sequence homology. The larger HMGs (1 and 2) have molecular weights of about 29 000, and the smaller HMGs (14 and 17) have molecular weights of about 10 000. The calf thymus HMG proteins and highly homologous proteins from other vertebrate animals have unusual amino acid compositions in that they are very highly charged, about 50%, and the charges are about evenly split between basic and acidic amino acid residues. The HMG proteins are major non-histone chromatin proteins in that they are about 3- 10% as abundant as histones in purified chromatin3. The HMG proteins have at- tracted much attention because of the evidence that HMGs 14 and 17 are structural proteins of transcriptionally active chromatin 4 - 6. Suggestions have also been made that HMGs 1 and 2 are involved in transcription, replication and nucleosome for- mation (reviewed in ref. 6).

Despite the intriguing speculations, the functions of HMG proteins are as yet unknown. Thus, the proteins cannot be defined on the basis of their biological roles. Instead, they are operationally defined as the proteins that are extracted from purified chromatin with 0.35 M sodium chloride and are soluble in 2% trichloroacetic acid’s’. This operational definition has been applied to chromatin proteins of a wide variety of vertebrate animals without ambiguity ‘. However, it is clear that HMG proteins have not been as evolutionarily conserved as histones, and the relationship of oper-

0021-9673/87/$03.50 0 1987 Elsevier Science Publishers B.V.

328 S. SPIKER, M. BATES, L. J. ARWOOD

ationally defined HMG proteins from other taxa to HMG proteins of vertebrate animals is not well established7. For example, wheat embryos have four chromosomal proteins that can be operationally defined as HMG proteins8sg. These wheat proteins have the hallmark amino acid compositions of HMG proteinsg, and they have several other traits in common with vertebrate animal HMG proteins, e.g. they are released from nuclei upon treatment with DNase Ilo, and they are found in the fraction of chromatin most readily solubilized by micrococcal nucleaseg. However, peptide maps of the wheat HMG proteins show no obvious similarities to those of vertebrate ani- mal HMGsg, and limited N-terminal sequencing has failed to reveal homologies’i.

In order to obtain milligram quantities of the wheat HMG proteins for further study, we wished to take advantage of the speed and resolving power of high-per- formance liquid chromatography (HPLC). Although several reports have appeared on the use of reversed-phase HPLC to fractionate vertebrate animal HMG pro- teins12-ig, none of the reversed-phase systems results in the satisfactory resolution of all four wheat HMG proteins. Therefore, we have employed a combination of weak cation-exchange and reversed-phase chromatography to achieve efficient separation of the wheat HMG proteins. The system can also be used to fractionate chicken erythrocyte HMG proteins.

MATERIALS AND METHODS

HMG proteins HMG proteins were isolated from dry wheat embryos as previously describedg.

HMG proteins were isolated from chicken erythrocytes essentially by the procedure of Rabbani et ~1.~~ as the proteins extracted from purified chromatin with 0.35 h4 sodium chloride and soluble in 2% trichloroacetic acid. Chicken erythrocytes were collected by the method of Rill et aL2* except that fresh blood was obtained from chickens with severed throats at a poultry processing plant and filtered through cheesecloth before centrifugation. Packed erythrocytes (40 ml) were ruptured in 200 ml of resuspension medium [0.5 M hexylene glycol, 10 mM 1,4_piperazinediethane- sulfonic acid (PIPES) pH 7.0, 10 mM magnesium chloride, 5 mM sodium chloride, 5 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride]. The nuclei were pelleted at 4000 g, washed twice in resuspension medium and washed an additional time in resuspension medium containing 0.5% Triton X-100. HMG proteins were isolated as previously described9 with a modification in the first sodium chloride extraction, Nuclei were suspended in 20 ml 75 mM sodium chloride, 25 mM NaED- TA pH 7.5 and then adjusted to 0.35 M sodium chloride by adding 0.7 M sodium chloride, 1 mM Tris-HCl pH 8.0 dropwise with brisk stirring of the sample.

Polyacrylamide gel electrophoresis Sodium dodecyl sulfate (SDS), polyacrylamide mini gels were run and stained

with Coomassie blue R-250 as previously describedz2.

High-performance liquid chromatography High-performance liquid chromatography was carried out on a Waters HPLC

gradient system including: 720 systems controller, 730 data module, WISP 710B sam- ple injector, Lambda Max 480 variable wave-length UV detector and two 6000A

FRACTlONATION OF HMG CHROMOSOMAL PROTEINS 329

solvent pumps. HPLC-grade water was obtained by use of a Millipore Milli-Q water purification system. HPLC-grade acetonitrile and phosphoric acid were obtained from Fisher. Reversed-phase chromatography was carried out on a Waters PBon- dapak C1 B column, and ion-exchange chromatography was carried out on a Brownlee Labs CX-300 weak cation-exchange guard column. Samples from ion-exchange chro- matography were desalted and concentrated by use of Amicon Centricon 10 micro- concentrators according to the manufacturer’s directions. Samples from reversed- phase chromatography were diluted with water and lyophilized before desalting.

RESULTS

Reversed-phase HPLC of wheat embryo HMG proteins Fig. 1 shows the elution profile of wheat HMG proteins separated by reversed-

phase HPLC. Fraction 1 is a doublet containing HMGb and HMGc. Fraction 2 of Fig. 1 contains HMGa and HMGd. Fig. 2 is an SDS gel of the fractions obtained in Fig. 1.

Reversed-phase HPLC of chicken erythrocyte HMG proteins Fig. 3 shows the elution profile of chicken HMG proteins (containing histone

H5 as a contaminant) separated by the same reversed-phase procedures as those used for the wheat HMG proteins. Chicken HMG14 and HMG17 (fraction 1) elute much earlier than any of the plant HMG proteins. Chicken HMGl, HMG2 and histone H5 elute at approximately the same positions as the wheat HMG proteins. Fig. 4 is an SDS gel of the fractions obtained in Fig. 3.

0’ lb 2b

Time fmin I

Fig, I. Reversed-phase HPLC of wheat embryo HMG proteins. Approximately 2 mg of a wheat HMG sample dissolved in 100 ~1 water was applied to the reversed-phase column and eluted at a flow-rate of 1 ml/min with the following gradient. From “inject” to 1 min: 100% buffer A (50 mM sodium phosphate, pH 3.0). From 1 min to 21 min: a linear gradient from 100% A to 100% B (50 mM sodium phosphate, pH 3.0, 60% acetonitrile). From 21 min to 26 min: 100% B. From 26 min to 27 min: a linear gradient from 100% B to 100% A. From 27 min to 32 min: 100% A. Elution was monitored by absorbance at 230 nm. The HMG-containing fractions labeled 1 and 2 were collected manually. Polyacrylamide gels shown in Fig. 2 indicate that the fraction 1 doublet contains HMGb and HMGc and fraction 2 contains HMGa and HMGd.

330 S. SPIKER, M. BATES, L. J. ARWOOD

Fig. 2. SDS polyacrylamide gel analysis of the fractions of wheat HMG proteins obtained by reversed- phase chromatography in Fig. 1. Lane 1 is the proteins contained in fraction 1 from Fig. 1. Lane 2 is the proteins contained in fraction 2 of Fig. 1. Between lanes I and 2 is a standard of unfractionated wheat HMG proteins. The positions of wheat HMGa, b, c and d are indicated on the left. The molecular weights of the wheat HMG proteins have been estimated to be HMGa: 23 100, HMGb: 14 500, HMGc: 10 000 and HMGd: 6300 using pig thymus HMG proteins as moIecular weight standardsQ.

“I

0 lb 2b Time tmin I

Fig. 3. Reversed-phase HPLC of chicken erythrocyte HMG proteins. Approximately 1 mg of a chicken HMG sample containing histone H5 as a contaminant was dissolved in 100 ~1 water and applied to the reversed-phase column. The elution gradient was the same as that outlined in the legend to Fig. 1. Fraction 1 contains chicken HMG14 and HMG17. Fraction 2 contains HMGI and fraction 3 contains HMG2 and histone H5.

FRACTIONATION OF HMG CHROMOSOMAL PROTEINS 331

Fig. 4. SDS polyacrylamide gel analysis of the chicken HMG proteins obtained by reversed-phase chro- matography in Fig. 3. Lanes l-3 are the proteins contained in fractions 1-3 (respectively) from Fig. 3. A lane of the, unfractionated proteins is shown on the left.

Weak cation-exchange HPLC of wheat embryo HMG proteins Because reversed-phase HPLC failed to give us clean separation of all four

wheat embryo HMG proteins, we turned to weak cation-exchange HPLC. Fig. 5 shows the resulting elution profile. The wheat HMG proteins are collected in two fractions (1 and 2), each of which contains two- peaks. The first fraction contains wheat HMGd in the first peak and HMGc in the second peak. Essentially pure HMGd can be obtained by collecting the leading edge ti peak 1 of fraction 1, and essentially pure HMGc can be obtained by collecting.the trailing edge of peak 2 of fraction 1. The second fraction contains HMGb and HMGa. Essentially pure HMGb can be obtained by collecting the leading edge of peak 1 of fraction 2, and essentially pure HMGa can be obtained by collecting the trailing edge of peak 2 of fraction 2. The unlabeled peak eluting last from the ion-exchange column contains Hl histones which frequently contaminate wheat HMG preparations. Fig. 6 is an SDS gel of the proteins collected in fraction 1 (both peaks) and fraction 2 (both peaks) in Fig. 5.

We have found that when we carry out repeated injections to isolate large quantities of protein, we obtain more consistent separations if, between protein in-

332 S. SPIKER, M. BATES, L. J. ARWOOD

Time (min I

Fig. 5. Weak cation-exchange HPLC of wheat embryo HMG proteins. Approximately 2 mg of a wheat embryo HMG sample dissolved in 100 ~1 water was applied to the weak cation-exchange column and &ted at a flow-rate of 1 ml/min with the following gradient. From “inject” to 5 min: 100% buffer A (50 mM potassium phosphate, pH 5.9). From 5 min to 46 min: a linear gradient from 100% A to 100% B (50 mM potassium phosphate pH 5.9, I M sodium chloride). From 45 min to 55 min: 100% B. From 55 min to 56 min: a linear gradient from 100% B to 100% A. From 56 min to 60 min: 100% A. Wheat HMG proteins were collected as fractions labeled 1 and 2. Fraction 1 is a doublet containing HMGd (peak 1) and HMGc (peak 2). Fraction 2 is also a doublet containing HMGb (peak 1) and HMGa (peak 2). The unlabeled peak eluting after the HMG proteins contains wheat Hl histones, which frequently contaminate HMG preparations. An SDS gel of the proteins from fractions I and 2 is shown in Fig. 6.

jections, we run a blank gradient (no protein injected) followed by one-half hour of washing the column in 100% A.

Weak cation-exchange HPLC of chicken erythrocyte HMG proteins A preparation of chicken HMG proteins, which contains histones Hla, Hlb

and H5 as contaminants, was separated by weak cation-exchange HPLC under the same conditions used to separate wheat HMG proteins. The elution profile is shown in Fig. 7, and SDS gels of the fractions collected are shown in Fig. 8. Chicken histone H5 and HMG2 elute at approximately 20 min, about the same position as wheat HMGc and HMGd. Chicken HMGl, HMG14 and HMG17 elute at about 25 min, which is very close to the position of elution of wheat HMGa and HMGb. Chicken Hl histones elute at about 35 min, somewhat earlier than the wheat Hl histones.

Isolation of all four wheat HMG proteins As mentioned above, all four wheat HMG proteins can be isolated in essen-

tially pure form by judicious collection of leading and trailing peaks from the ion- exchange columns. This procedure results in considerable reduction in yield. By com- bining the procedures of reversed-phase and ion-exchange HPLC the wheat HMG proteins can be obtained in essentially pure form at nearly 100% yields. Our routine procedure is to first subject the wheat HMG proteins to ion-exchange chromato- graphy and collect HMGc and HMGd in fraction 1 and HMGa and HMGb in fraction 2, as shown in Figs. 5 and 6. Following collection, the fractions are desalted and concentrated by use of Centricons. The resulting fractions are separately sub- jected to reversed-phase HPLC. Each HPLC system is capable of resolving two frac- tions containing two HMG proteins. Because different pairs of proteins cofractionate

FRACTIONATION OF HMG CHROMOSOMAL PROTEINS 333

Fig. 6. SDS polyacrylamide gel analysis of the wheat embryo HMG proteins obtained by weak cation- exchange HPLC in Fig. 5. Lane 1 contains the proteins found in fraction 1 of Fig. 5. Lane 2 contains the proteins found in fraction 2 of Fig. 5. The positions of wheat HMGaad are indicated on the left.

1.0

8 P I 1 I73

II

I

1’1

0’ I I

20 40 Time (min )

Fig. 7. Weak cation-exchange HPLC of chicken erythrocyte HMG proteins. Approximately I mg of a chicken HMG sample was dissolved in 100 ~1 water and applied to the weak cation-exchange column. The ehrtion was exactly the same as that outlined in the legend to Fig. 5. The chicken HMG preparation is heavily contaminated by histones Hla, Hlb and HS. The proteins eluted are collected in four fractions. Fraction 1 contains HMG2 and histone H5. Fraction 2 contains HMGt. Fraction 3 contains HMG14 and HMG17. Fraction 4 contains histones Hla and Hlb.

S. SPIKER, M. BATES, L. J. ARWOOD

Fig. 8. SDS polyacrylamide gel analysis of the ohicken erythrocyte proteins obtained by weak cation- exchange HPLC in Fig. 7. Each numbered lane corresponds to the proteins of that fraction from Fig. 7. Lane 1 contains HMG2 and histone H5. Lane 2 contains HMGl. Lane 3 contains HMG14 and HMG17. Lane 4 contains histones Hla and HI b. The lane on the far left contains the unfractionated HMG prep- aration applied to the column in Fig. 7. -Positions of histone HI (top band is Hla and bottom band is Hlb), histone H5 and HMGs 1. 2, 14 and 17 are indicated.

in each system, combining the”two systems results in clean separation of all four proteins with very little loss of protein. An SDS gel of wheat HMG fractions purified by this procedure is shown in Fig. 9.

DISCUSSION

We have devised a combination of reversed-phase and weak cation-exchange HPLC systems to enable us to efficiently obtain wheat HMG proteins of high purity. The proteins obtained are free of extraneous bands even on heavily overloaded SDS gels. The use of weak cation-exchange HPLC by itself can result in proteins of nearly the same purity but at much lower recovery. We have also tried strong cation-ex- change HPLC, similar to that employed by Riffe et dz3. These procedures did not result in a useful separation of the wheat HMG proteins.

Wheat HMGb, HMGc and HMGd purified by the combination weak cat- ion-exchange and reversed-phase systems all have single N-terminal amino acids and

FRACTIONATION OF HMG CHROMOSOMAL PROTEINS 335

Fig. 9. SDS polyacrylamide gel analysis of the wheat embryo HMG proteins obtained by use of ion- exchange HPLC followed by reversed-phase HPLC. Wheat embryo HMG proteins were separated into frachon I and fraction 2 by ion-exchange chromatography. After desalting and concentrating, the proteins in each fraction were then further separated by reversed-phase HPLC. The proteins resulting from this fractionation were then run on SDS polyacrylamide gels. Lanes 3, 6 and 9 are standards of unfractionated wheat embryo HMG proteins. The positions of HMG proteins a, b, c and d are shown on the left. Lanes I and 2 contain HMGa. Lanes 4 and 5 contain HMGb. Lane 7 contains HMGc, and lane 10 contains HMGd.

yield unique primary structures as far as they have been sequenced by automated Edman degradation (up to at least I8 residues and more than 30 in some cases, data not shown). HMGa has not yet been investigated.

The weak cation-exchange system can be used to effectively purify chicken HMGl and HMG2 (but not subfractions of HMG2). HMG14 and HMG17 are eluted essentially together. Thus the ion-exchange system is not as effective as the reversed-phase system previously reported for fractionation of vertebrate animal HMG proteins 12-lg. However, it is possible that proteins separated by cation-ex- change chromatography may be more suitable for some biochemical and biophysical studies than proteins fractionated by reversed-phase chromatography.

ACKNOWLEDGEMENTS

This work was supported in part by National Science Foundation Grant DCB-84093 14. We thank Keith Everett for technical work. Wheat germ was provided by General MilIs, Minneapolis, MN and chicken blood was provided by Goldkist Poultry, Durham, NC, U.S.A. This is paper 11055 of the Journal Series of the North Carolina Agricultural Research Service.

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336 S. SPIKER, M. BATES, L. J. ARWOOD

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