Purification of coniferyl alcohol oxidase from lignifying xylem of sitka spruce using immobilised metal affinity chromatography

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© 1998 by Gustav Fischer Verlag. Jena

_Purification of Coniferyl Alcohol Oxidase from Lignifying Xylem of Sitka Spruce Using Immobilised Metal Affinity Chromatography GORDON

J. McDOUGALL

Unit of Plant Biochemistry. Cellular and Environmental Physiology Department. Scottish Crop Research Institute, Invergowrie Dundee, DD2 5DA, United Kingdom

Received February 27,1998· Accepted March 31, 1998

Summary Extracts of lignifying xylem of Sitka spruce, obtained by a combined extraction/affinity method that selects for cell-wall-associated glycoproteins, were enriched in oxidase and peroxidase activity but peroxidase actiyity was approximately 80 times more abundant than oxidase activity. Previous attempts to purify the oxidase using ion-exchange, hydrophobic interaction and gel filtration chromatographic methods have been unsuccessful and it has proved patticularly difficult to separate completely oxidase activity from peroxidase activity. All of the oxidase activity in the xylem cell wall extracts bound to an affinity matrix loaded with immobilised copper (Cu2+) ions and was fractionated into two main peaks by eluting the bound proteins with a gradient of histidine. The second oxidase peak, which represents a subset of oxidase that has higher affinity for the bound metal ions, contained a third of the total oxidase activity at a 2-fold increase in specific activity. In addition, this fraction had gready reduced although still detectable peroxidase activity. Oxidase activity was split into a bound and an unbound fraction when extracts were applied to a matrix of immobilised cobalt (Co2+) ions. In this case. a larger proportion of the applied oxidase activity than peroxidase activity bound to the Co2+ -loaded matrix and the specific activity was higher than that in the unbound fraction. This suggested that this bound fraction of the oxidase population had a higher affinity for metal ions and could therefore be isolated from peroxidase. When applied to a Zn2+ -loaded matrix. the oxidase activity also split into bound and unbound portions but effectively all of the peroxidase activity was eluted in the unbound fraction yielding a bound fraction enriched in oxidase activity with negligible peroxidase contamination. The nature of this «high affinity fraction» of oxidase and the heterogeneity in the oxidase population is discussed. Gel permeation on Superose-6 partially resolved oxidase from the residual peroxidase activity in the Znbound fraction and gave an apparent Mr of 62 kDa for the oxidase activity. However. an examination of the protein profile of fractions enriched in oxidase activity by SDS-PAGE strongly suggests that a protein band of apparent Mr 80 kDa is responsible for the oxidase activity. This apparent disparity is discussed. In summary. immobilised metal affinity chromatography on Zn-Ioaded matrix provides a rapid method to separate oxidase from peroxidase activities enabling studies of the substrate specificity and the physical properties of the oxidase to be carried out.

Key words: Picea sitchensis. lignification. xylem development. cell wall, oxidase. coniflryl alcohol purification, immobilised metal affinity chromatography. ]. Plant PhysioL WlL 153. pp. 539-544 (1998)

540

GORDON

J. MCDOUGALL

Abbreviations: ABTS = 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid); CA = coniferyl alcohol; IMAC = immobilised metal affinity chromatography; PVP = polyvinylpyrrolidone; PMSF = phenyl methyl sulphonyl fluoride. Introduction

We have previously shown that the lignifying, differentiating xylem of Sitka spruce contains a cell-wall-associated oxidase activity that can oxidise and polymerise coniferyl alcohol to form lignin-like material (McDougall and Morrison, 1996; Richardson et al., 1997). To define properly the biochemical and kinetic properties of the oxidase e.g. substrate specificity, product formation, K.n, Vmax etc., the oxidase should be purified or, at least, separated from contaminating activities such as peroxidase. Complete separation of oxidase from peroxidase activity is crucial as peroxidases, particularly when present at high concentrations, can catalyse certain oxidation reactions in the absence of hydrogen peroxide (Gaspar et al., 1982; Kanofsky, 1991). Chromatographic techniques, such as ion exchange, hydrophobic interaction and gel permeation chromatography (and combination of these methods), that have been successfully employed by other researchers to separate and purify oxidases (Sterjiades et al., 1992; Bao et al., 1993; Chabanet et al., 1994), do not effectively separate oxidase from peroxidases in extracts of developing xylem of Sitka spruce. For example, gel permeation chromatography provided only partial resolution of oxidase and peroxidase activities in extracts of lignifying xylem of Leyland cypress (Richardson and McDougall, 1998) and Sitka spruce (results not shown). Hydrophobic interaction chromatography on Phenyl Sepharose was also ineffective (results not shown) and, as observed before for tobacco xylem oxidases (Richardson and McDougall, 1997), oxidase activity tended to precipitate in the concentrated ammonium sulphate buffers required by this technique. Ion exchange chromatography on DEAESepharose provided the best separation of oxidase and peroxidase activity from Sitka xylem extracts. The most acidic oxidase isoform was resolved from the main isoforms of peroxidase but still had appreciable peroxidase contamination (Richardson et al., 1997). Therefore, oxidase and peroxidase are both glycoproteins (Richardson et al., 1997), have similar and overlapping isoform profiles and lack apparently exploitable differences in hydrophobicity. Immobilised metal affinity chromatography (IMAC) takes advantage of the interactions between available charged sidechains of amino acids, such as histidine, tryptophan and cysteine, on the surface of proteins and transition metal ions chelated via iminodiacetate groups to a matrix (Ostrave and Weiss, 1990). The number, type and arrangement of charged groups influences the strength of binding. In addition, different groups can have very different affinities for different metal ions but, generally speaking, proteins bind more strongly to Cu2+ than Zn2+ ions (Porath, 1992). As the binding of proteins can also be modulated by the inclusion of counter ions or by altering the pH of the loading buffer and proteins can be selectively desorbed by pH changes, competitive ligands (e.g. histidine) or gradients of salt or chelating agents (Arnold, 1991), IMAC is very versatile and effective in resolving proteins with similar properties. In this paper, a

simple and gentle IMAC method is reported for the separation of oxidase from peroxidase in extracts of cell-wall-associated proteins of Sitka spruce xylem.

Materials and Methods

Plant matmal Branches of Sitka spruce (Picea sitchmsis ([Bong] Carr.) were obtained from a genetically defined clone at the Forest Authority tree bank at Ledmore, Perthshire, UK, in mid-May 1997 when oxidase activity against ABTS was high. The branches (minimum 5 cm in diameter) were removed to the laboratory and the bark peeled away to reveal the cambial tissue. The milky-white cambial tissue was scraped from the wood using a razor blade and stored at -20 'c.

Extraction ofceil-wail-associated proteins and Concanavalin-A affinity chromatography Developing xylem (400 g fresh weight) was thawed and suspended in ice-cold 25 mmollL MOPS pH 7.0 containing 0.25 % (w/v) polyvinylpyrrolidone (PVP) and 0.5 mmol/L phenyl methyl sulphonyl fluoride (PMSF). The tissue was first homogenized using a Waring Blender (5 X 30 sec bursts, cooled on ice) then using an Ultra-Turrax disintegrator (10 X 30 sec bursts, full power, cooled on ice). The insoluble material was collected by filtration through muslin and the filtrate collected as the soluble fraction. The insoluble material was then repeatedly homogenized in 25 mmollL MOPS pH 7.0 until the filtrates were no longer coloured. Afrer a final wash with buffer, the crude cell wall preparations were extracted with 25 mmollL MOPS pH 7.0 containing 200 mmollL CaCl2 (ConA buffer) for 1 hour on ice. The extracts were then collected by filtration through muslin and clarified by passage through Whatman GFI A glass fibre filters. The ConA buffer extract was loaded onto a 15 mL bed volume column of Concanavalin-A Sepharose (Pharmacia Ltd., St. Albans, UK), that had been loaded with 1 mmollL MnCl2 then pre-equilibrated in ConA buffer. Afrer all the ConA extract was applied, the column was washed with 25 mL of ConA buffer then eluted with 15 mL ConA buffer containing 100 mmollL a-methyl mannoside. The entire process was carried out at 8 'C and eluates from each step were collected, assayed for protein content and peroxidase and oxidase activity. When required, samples were concentrated using Centriplus-50 centrifugal membrane units as described by the manufacturer (Amicon Ltd., Stonehouse, UK).

Peroxidase, oxidase and coniforyl alcohol oxidase assays For general oxidase assays, ABTS was used at a final concentration of 1.82 mmollL in 100 mmollL acetate buffer pH 5.0. The rate of production of the coloured ABTS chromophore [E420 = 3.6 X 10- 4 M-i cm-i (Sterjiades et al., 1992)] was measured. Peroxidase assays were similar but 0.5 mmollL H 20 2 was added. Coniferyl alcohol oxidase (CAO) activity was measured by the rate of decline in absorbance at 260 nm (A260) using 0.2 mmollL coniferyl alcohol (CA) in 100 mmollL acetate buffer pH 5.0. The extinction coefficient of CA was estimated empirically. Protein concentration was measured by the method of Bradford (1976).

Purification of Coniferyl Alcohol Oxidase

Analytical SDS-PAGE This technique was carried out on 9.5 % polyacrylamide gels using a Mini-Protean II slab gel system according to the makers instructions (Bio-Rad Ltd., Hemel Hempstead, UK). Standard protein markers for molecular weight estimation were obtained from BioRad Ltd. (low molecular weight range) and gels were stained using the Bio-Rad silver stain method. Proteins in samples were routinely precipitated by the addition of a two-fold excess of cold (-20°C) acetone, stored overnight at -20°C and collected by centrifugation. The precipitated proteins were then re-dissolved in SOS-PAGE sample buffer.

3.0

Gel permeation chromatography on Superose-6 The Superose-6 FPLC column was pre-equilibrated in ConA buffer at 0.25 mUmin. The sample (200 ilL) was loaded and the column eluted with the same buffer. The absorbance of the eluate was monitored at 280 nm and fractions (0.5 mL) were collected and assayed for oxidase and peroxidase activity. Fractions containing oxidase activity were pooled as shown. Standard proteins (low molecular tange, Pharmacia LKB Biotechnology Ltd.) were run and their elution times used to construct a standard curve for estimating molecular weight.

Results

Oxidase and peroxidase act!vltles were extracted from xylem cell walls using buffered 200 mmol/L CaCl2 but peroxidase activity was 80-fold more abundant (Table 1). Only approx. 50 % of the total oxidase and peroxidase activity bound to Concanavalin-A affinity columns and was eluted using a-methyl mannoside. However, some of the unbound oxidase activity could subsequently be recaptured by binding to Concanavalin-A suggesting that the available lectin groups of the original column were saturated. The ConA bound fraction was enriched 4.2-fold in oxidase activity but, as this method selects for cell-wall-associated glycoproteins (McDougall, 1997), peroxidase activity was also enriched 3.0-fold. The ConA bound fraction was routinely concentrated by centrifugal membrane filter units with nominal cut-offs at 50 kDa. Concentrates retained all of the applied oxidase but

... 2.0

2.0 0-<> 1.0

Immobilised metal affinity chromatography A Chelating Superose FPLC column attached to an FPLC system was used according to the manufacturers instructions (Pharmacia LKB Biotechnology Ltd., St Albans UK). Before use and between changes of metal ions, the column was cleaned and stripped of metals by washing with 50 mmollL sodium EOTA in 500 mmollL NaCl. The column was washed with excess water, loaded with 100 mmollL solutions of the metal salt of choice (CuCh, CoCh and ZnS04), washed again with excess water then pre-equilibrated in buffer A (25mmollL MOPS pH 7.0 containing 50mmollL CaCh). The sample was loaded and eluted with buffer A for 20 min at 1 mUmin then bound proteins were eluted using a gradient of 0-100 % buffer B (25 mmollL MOPS pH 7.0, 50 mmollL CaCh containing 25 mmollL histidine) over 50 min then held at 100 % for a further 10 min. Fractions (1 mL) were collected and assayed for oxidase and peroxidase activity. Fractions containing oxidase activity were pooled as shown in Fig. 1. Fractions were desalted into deionised water prior to storage by passage through PO-IO desalting columns (Pharmacia LKB Biotechnology Ltd.).

541

1.0

o

2.0

1.0

o 3.0

c

2..0

1.0

o Volume(mQ Fig. 1: Affinity of oxidase for different chelated metal ions Oxidase activity (e) and peroxidase activity (<» were measured in fractions from the IMAC column loaded with Cu2 + ions (Fig. 1 a), C02 + ions (Fig. 1 b) and Zn2+ ions (Fig. 1 c). The gradient of histidine (025 mmollL) is shown and the oxidase fractions were pooled as shown.

caused a slight decrease in oxidase specific activity and increase in peroxidase specific activity (Table 1). Oxidase activity against coniferyl alcohol in these fractions closely mirrored ABTS oxidase activity but the specific activity against CA was always 2.5 - 3 fold higher.

542

GORDON

J. MCDOUGALL

Table 1: Enrichment of oxidase and peroxidase activity by Concanavalin A affinity chromatography and centrifugal filtration. OXIDASE

Specific Activity'

Total Activity'

593±27

18570±550

323120±1497 [306410]

2051±67 [1618]C

3214±74

55660± 1278

170140±667 [161630]

1838±20

2086±58

59937±333

163659 ± 1195

Specific Activity'

Total Activity'

Cell wall extract

233±15

4031 ±33 [3821]C

Concanavalin-A Bound Fraction

974±18

Concanavalin-A Concentrate I

673±14

Sample

PEROXIDASE

CAOXIDASE Specific Activityb

a-specific activity = nmols ABTS min -\ mg-I , total activity = nmols ABTS min -\ . b-specific activity = nmols CA min -\ mg -I. c-figures in square brackets represent the amount of activity taken on to the next step.

Table 2: Affinity of oxidase and peroxidase activitis for different chelated metals. PEROXIDASE

OXIDASE Specific Activity'

Total Activity'

Specific Activity'

Total Activity'

Ratio b

ConAconc I Cu-bound I Cu-bound II

672±12 1192±25 1424±14

1838±20 792±20 617± 14

59937±333 27717±278 16263±695

163659±1195 18181±556 6978±389

0.010 0.043 0.088

ConAconc II Co-unbound Co-bound

751±36 581±17 620±8

19lO±17 731±16 211±3

61994±389 58519±1278 16541±167

153233±361 72836±501 5645±256

0.012 O.OlO 0.037

ConA conc III Zn-unbound Zn-bound

881±16 756±14 1195±55

93658± 1584 212169±2418 5282±417

2930±80

197853±1863 152761 ±2085 1056±28 [201]C 48±2

0.010 0.003 0.226

Zn-boundGF

1863± 14 545±8 293±3 [56]C 32±1

Sample

2448±202

1.197

a-specific activity = nmol ABTS min -I mg -I protein. total activity = nmols ABTS min -I . b-ratio of the oxidase over the peroxidase specific activities of the samples. c-figures in square brackets represent the amount of activity taken on to the next step.

All of the oxidase in the concentrated ConA bound sample bound to the Cu2 +-loaded IMAC column and was separated into two main fractions by a gradient of histidine (Fig. 1 a). The first fraction (Cu-bound I) contained 43 % of applied oxidase at 1.8-fold purification with respect to the applied sample (Table 2). The second fraction (Cu-bound II) contained 33 % of applied oxidase activity at 2.1-fold purification. Both peaks contained peroxidase activity (11 and 4 % total respectively.) but at lower purity (0.46 and O.27-fold respectively) than the applied sample (Table2). The ConA bound concentrate gave an unbound and a bound fraction of oxidase activity when applied to the lMAC column that had been loaded with C0 2 + ions (Fig. 1 b). Neither fraction was entiched in oxidase w.r.t. the original sample (0.77 and 0.82-fold and 38 % and 11 % total respectively.) but the Co-bound fraction was particularly reduced in peroxidase activity (0.27-fold and 3.7% total) (Table 2). This indicates that there was a subset of the oxidase population that has a higher affinity for the immobilised metal ion than the peroxidase population. The concentrated ConA bound sample also gave an unbound and a bound fraction of oxidase when applied to the Zn2 +-loaded IMAC column (Fig. I c). The Zn-unbound fraction was not enriched in oxidase (0.86-fold and 29 % to-

tal) but it was enriched in peroxidase activity (2.27-fold and 77 % total) compared to the original sample (Table 2). On the other hand, although the Zn-bound fraction represented a comparatively low yield of oxidase (15.7%), it was enriched in oxidase activity (I.4-fold) and it had negligible peroxidase activity (O.06-fold and 0.5 % total) (Table 2). The residual peroxidase results from carry-over from the unbound fraction as it can be removed by increasing the wash time afrer the application of sample and before the histidine gradient (results not shown). Although oxidase activity was not inhibited by the addition of 1 mmol/L Zn2 +, C0 2 + or Cu2+ ions (Richardson et al., 1997), these ions are present at 5-10 mmollL in the bound fractions and they may cause inhibition. Such inhibition may explain the low yield of oxidase in the Co- and Znbound fractions. Indeed. these ions did affect oxidase stability and extracts were desalted prior to storage. Oxidase and residual peroxidase in the Zn-bound fraction were partially resolved by gel permeation on a Superose-6 column (Fig. 2). The oxidase activity eluted first at an apparent Mr of 62 ± 12 kDa and was followed closely by the perOludase at an apparent Mr of 42 ± 12 kDa. The oxidase containing fractions from this step were pooled (Zn-bound GF, see shaded area, Fig. 2) and this sample was enriched in oxidase

Purification of Coniferyl Alcohol Oxidase

A280

A420

0.06

.&~~--------------------------,

0.04

0.6

0.02

0.4

o

0.2

o

543

However, when more protein from the Zn-bound fraction was applied to 50S-PAGE, more bands became apparent including the probable source of the peroxidase contamination at 38-40 kDa (Fig. 4, lane C). Nevertheless, the Zn-bound GF fraction contained three protein bands at approx. 80, 65 and 60 kDa of which only the 80 kDa band was enriched over the Zn-bound fraction (Fig. 4, compare lanes C & 0). This indicates that the 80 kDa protein band may be responsible for the oxidase activity. The Mr of this band is similar to the Mr of the oxidase band (84 kDa) identified previously using activity staining and non-denaturing 50S PAGE of xylem extracts (Richardson et al., 1997). Discussion

20

10

Elution Volume(mQ

Fig. 2: Separation of oxidase and peroxidase activities by gel permeation chromatography. Oxidase activity (e) and peroxidase activity (0) were measured in fractions from the Superose-6 gel permeation column. The trace of absorbance at 280 nm is also shown and is displaced for clarity.

ABCDEFG

I


--

Fig. 3: Separation of proteins in IMAC fractions by SDS-PAGE Lanes A - F contain 2. 511g protein and lane G contains 1l1g protein. Lane A =ConA cone. I, lane B = Cu-bound I, lane C = Cu-bound II, lane D = Co-unbound, lane E = Co-bound, lane F = Znunbound and lane G = Zn-bound. The positions of molecular weight markers (97.4, 66.2, 45 and 30 kDa) are denoted by closed arrows and the putative oxidase band by the open arrow.

activity (2.45-fold and 57% total) but depleted in peroxidase activity (0.46-fold and 23 % total) (Table 2). By comparing the 50S-PAGE protein profiles of fractions that are enriched in oxidase activity, one can ascertain which protein bands may be responsible for the oxidase activity. For example, the Cu-bound II fraction, which had the greatest enrichment of oxidase activity amongst the lMAC fractions, contained only six main bands (Fig. 3, lane C). However, the Zn-bound fraction, which was also enriched in oxidase, contained only three bands at approx. 80, 65 and 60 kDa (Fig. 3, lane G) and neither the 65 nor the 60 kDa bands appear to have to been enriched compared to the Zn unbound fraction.

The combined extraction/Concanavalin-A affinity procedure provided a convenient means to enrich oxidase activity from the dilute protein/high volume extracts of the cell walls of developing xylem but it has the disadvantage that it also enriches other cell-wall-associated glycoproteins, in particular peroxidase (Table 1). Oxidase and peroxidase could be split into two main fractions by their affinity for immobilised copper, cobalt or zinc ions and the fraction that bound more strongly to the metal ions generally had higher oxidase and a higher oxidase/peroxidase ratio. Exploitation of the general lower affinity of proteins for chelated Zn2 + ions (Porath, 1991) allowed the separation of a high affinity oxidase fraction which was free of peroxidase. This subset of oxidases may differ in either the number, type or arrangement of accessible surface charged groups (Hemdan et al., 1989). However, the high and lower affinity oxidase fractions do not represent different types of oxidases with different specificities as these fractions share common substrate and inhibitor profiles and are equally capable of oxidising coniferyl alcohol (results not shown). Theoretically, the acidic oxidase isoforms that were partially separated from peroxidase isoforms by ion-

A B C 0

Fig.4: Separation of proteins from fractions by SDS-PAGE. Each lane contains 2.5l1g protein. Lane A =ConA cone III, lane B = Znunbound, lane C = Zn-bound and lane D = Zn-bound GF oxidase peak sample. The positions of molecular weight markers (97.4, 66.2, 45 and 30 kDa) are denoted by closed arrows and the putative oxidase band by the open arrow.

544

GORDON J. MCDOUGALL

exchange chromatography on DEAE~Sepharose (Richardson et aI., 1997) would have more negative charges at neutral pH and bind more strongly to immobilised metal ions. However, attempts to confirm differences in the isoform composition of oxidases of the Zn~bound and unbound fractions using isoelectric focusing have not been successful. Differences in the accessibility or arrangement of charged surface groups could result from microheterogeneity in protein sequence be~ tween oxidase isoforms [as described for Liriodendron laccases (LaFayette and Dean, 1997)] or from microheterogeneity in glycosylation [as described for Acer laccases (Tezuka et aI., 1993)]. However, the nature of the difference between the high and low affinity oxidase fractions remains to be discov~ ered. The apparent disparity between the molecular weights esti~ mated for the oxidase by SDS~PAGE (-80 kDa) and gel per~ meation chromatography (-60 kDa) may arise because the oxidase is a glycoprotein. Glycosyl chains of glycoproteins do not bind SDS and interfere with the binding of SDS by the protein chain (Hames and Rickwood, 1981). Therefore, gly~ coproteins tend to carry less charge per unit mass, migrate less upon SDS~PAGE electrophoresis and their molecular weights can be over~estimated. However, it is noteworthy that the oxidase band at 80 kDa identified in this paper has a sim~ ilar Mr to the 84 kDa oxidase band identified by activity staining after non-denaturing SDS~PAGE (Richardson at aI., 1997). The oxidase from lignifying Sitka xylem has a considerably lower Mr than reported for other oxidases from developing tree xylem. For example, the oxidase from Acer pseudoplatanus had a Mr of 97kDa estimated by SDS~PAGE (Sterjiades et aI., 1992), the oxidase from Pinus tadea had a Mr of 90 kDa estimated by SDS~PAGE but the deglycosylated enzyme was only 70 kDa (Bao et aI., 1993) and the oxidase from Pinus strobus had a Mr of 107kDa estimated by SDS~PAGE but the deglycosylated enzyme was only 67 kDa (Udagama-Rande~ niya and Savidge, 1995). Although these differences could be due to differential glycosylation or processing of a common precursor [and the IMAC technique may have selected a variant form of oxidaseJ, the Sitka enzyme also has a different substrate and inhibitor profile (Richardson et aI., 1997) and may represent a different type of enzyme. Nevertheless, con~ firmation of similarity will require the acquisition and com~ parison of sequence data. In summary, IMAC provides a simple and gentle proce~ dure for the enrichment and purification of oxidase that can be easily scaled up to provide sufficient oxidase for detailed substrate specificity assays and amino~terminal protein se~ quencing. Acknowledgements

Dr. Gary Lyon and Dr. Heather Ross are thanked for FPLC advice and facilities and Dr. Ian Morrison for help with the manuscript. I thank Dr. Steve Lee, Forest Research for access to the Sitka clone bank, and Andrew Richardson and Julie Duncan for technical and manual help. SCRI recieves grant~in~aid from The Scottish Of~ fice Agriculture, Environment and Fisheries Department.

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