Isoelectric focusing electrophoresis of lignin

ANALYTICAL

BIOCHEMISTRY

197,101-103

(1991)

Isoelectric Focusing Electrophoresis Marja-Leena VTT,

Received

Niku-Paavola

Biotechnical

January

Laboratory,

P.O. Box 202, SF-02151

Espoo, Finland

17,199l

Isoelectric focusing is introduced as a technique for the analysis of macromolecular lignin. The analysis is performed in a pH gradient from 3.6 to 10. Separated lignin fragments are visualized under uv light or by silver staining. The method can be used to distinguish between differently processed lignin preparations and to identify their components. Even the slight modification resulting from attack by ligninolytic enzymes could be detected. Q 1991 Academic press, ~nc.

Lignin is conventionally analyzed using the methods of organic chemistry and sophisticated equipment. Electrophoresis, known to be convenient for other natural macromolecules, has been used in only a few cases. The reason for this is the poor solubility of macromolecular lignin in buffers used in electrophoresis. Hitherto, monomeric phenols have been analyzed by paper electrophoresis (1) and immunoelectrophoresis is used for macromolecular lignosulfonates (2). In this work isoelectric focusing electrophoresis was adapted for the analysis of the alkaline-soluble fraction of macromolecular wood lignin. Although the lignin is modified during the solubilization and electrophoresis, the aim was to determine whether this method could be used to distinguish between differently processed lignin preparations. The method was tested using mechanically, chemically, and enzymatically modified wood lignin as well as monomeric and dimeric models for lignin substructures. MATERIALS

AND

METHODS

Lignin Preparations: Models for Lignin Substructures The macromolecular substances used were Kraft pine lignin (waste lignin from a sulfate process; Indulin, AT West Waco), lignosulfonates (waste lignin from a sulfite process; G. A. Serlachius Co., Finland), and milled wood

1 Financial ment Centre,

of Lignin’

support Finland

was obtained (TEKES).

from

ooo3-2697/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

The

Technology

Develop-

lignin (MWL)2 (3) prepared by B. Hortling (Finnish Pulp and Paper Research Institute, P.O. Box 136, SF00101 Helsinki, Finland). Guaiacol (Merck), veratryl alcohol (Fluka), and veratraldehyde (Fluka) were used as monomeric models for lignin substructures. A dimeric lignin substructure model, syringaresinol, was kindly provided by Professor L. A. Golovleva (Pushchino, USSR Academy of Sciences). IEF Analysis IEF was performed using an LKB 2117-003 Multiphor II Electrofocusing Unit (LKB, Sweden) according to the manufacturer’s instructions. Ultrathin (0.5 mm) gels were casted on LKB Gel Bond PAG films using an LKB 2217-200 UltroMould Gel Casting Unit. The gels were prepared using analytical-grade reagents (Merck). The total acrylamide concentration was 7.5% (w/v) and the degree of crosslinking 3% (obtained by diluting 29.1% acrylamide and 0.9% N,N’-methylene bis-acrylamide stock). The gels contained 1.6% (v/v) ampholytes (LKB Ampholine mixture for pH 3.5lo), 0.1% (v/v) N,N,N’JV’-tetramethylethylenediamine and 0.1% (w/v) ammonium persulfate. After polymerization the gel was assembled horizontally on the cooling plate (+4’C) of the Multiphor II. The temperature was maintained with a Lauda MGW cooling system (RCS thermostat and RC 6 water bath, Germany). Filter paper strips soaked in 1 M NaOH and 1 M H,PO, were placed at the cathodic and anodic ends of the gel, respectively. The prerun was performed for 10 min using a Pharmacia ECPS 3000/150 power supply (Sweden) set at 2000 V, 50 mA, and 25 W. Samples, dissolved in 0.2 N NaOH (10 mg/ml), were applied in lo- to 40-~1 aliquots on filter paper pieces at the surface of the cathodic end of the gel. The samples were run into the gel using the preliminary power setting for 20 min, after which the sample application papers were

* Abbreviations used: IEF, isoelectric dase; MnP, Mn-dependent peroxidase; PAGE, polyacrylamide gel electrophoresis; wood.

focusing; Lip, lignin peroxiMWL, milled wood lignin; PGW, pressurized ground

101

102

MARJA-LEENA

NIKU-PAAVOLA

and the other was dissolved used for IEF analysis.

Infrared

in 250 ~1 of 0.2

N

NaOH

and

Analysis

Analyses were performed on the freeze-dried samples using the KBr pellet method (1 mg sample/300 mg KBr). Spectra were recorded on a Perkin-Elmer FTIR 1760 spectrophotometer. RESULTS 12

3

4

567

FIG. 1. IEF of alkaline-soluble lignin samples: 1, Kraft lignin; 2, MWL; 3, lignosulfonate; 4, syringaresinol; 5, veratraldehyde; 6, veratry1 alcohol; 7, guaiacol. Polyacrylamide gel in an ampholine mixture, pH 3.5-10, viewed under uv. The pH gradient formed is indicated to the left. The arrowhead indicates sample application.

removed and the electrophoresis was continued for 30 min. After focusing, the pH along the gel surface was measured using a surface electrode (Ingold, Switzerland). Plates were viewed under uv light (302 nm) and photographed (Polaroid type 665, land film). The plates were fixed for 20 min in 20% (w/v) trichloroacetic acid to wash out the ampholytes, after which silver staining (4) was performed using a Bio-Rad Kit.

Modification

of Lignin

by Enzymes

A macromolecular lignin substrate, pressurized ground wood (PGW), was prepared from spruce wood according to the standard laboratory methods of the Finnish Pulp and Paper Research Institute. Lignin peroxidase (isoenzyme LiP 3) and Mn-dependent peroxidase (MnP) were purified from culture filtrate of the white rot fungus Phlebia radiata Fr. 79 (ATCC 64658) as described previously (5). PGW, 2 g dry weight, was suspended in 30 ml of 0.1 M sodium acetate buffer, pH 5. Purified lignin peroxidase (isoenzyme LiP 3, 110 nkat) and 4.4 pmol H,O, were added when the effects of LiP were investigated. In the case of MnP the incubation mixture was prepared similarly but also included 6 pmol MnSO,. Incubation was performed at room temperature for 24 h. Corresponding controls without enzymes were also prepared. The buffer-soluble fraction from each sample was separated by centrifugation and the insoluble residue was washed with buffer and water and dissolved in 30 ml of 0.2 N NaOH. After 48 h at room temperature the alkaline-soluble fraction was separated and divided into two 15-ml parts. The pH of the solutions was adjusted to 2.0 with concentrated HCl and the precipitates formed were collected. One lot was freeze-dried for ir analysis

AND

DISCUSSION

The separation of macromolecular lignin and lignin substructure models by IEF is shown in Figs. 1 and 2. Depending on the sample, visualization was possible either by viewing under uv (Fig. 1) or using silver staining (Fig. 2). Fractionation succeeded only in the pH gradient: on ordinary PAGE with homogeneous pH the lignin formed a single distinct band (results not shown). The fractionation seen in IEF is obviously a consequence of the electrophoretic mobility but also represents stepwise precipitation of lignin fragments at the decreasing pH values of the gradient. Fractions are seen only at pH values below 8.0. In addition to pH, characteristics of the ampholytes present appeared to play a role in the separation. Resolution using the Ampholine mixture, pH 3.5-10, was superior to that obtained with the other commercial ampholyte mixtures tested (Pharmalyte, pH 3-10; Ampholine, pH 5-8; and Ampholine, pH 3.5-5; results not shown). These ampholytes are synthetic mixtures of undefined polycarboxy-polyamine compounds optimized to maintain a stable pH gradient under the desired pH range. Obviously the ampholyte mixtures, depending on their composition, interract individually with the reactive groups of lignin fragments. The IEF analysis is suitable for the analysis of macromolecular lignin. Of the monomeric and dimeric models for lignin substructures only guaiacol was visualized and seen to move in electrophoresis (Figs. 1 and 2, lane 7). Kraft lignin, MWL, lignosulfonates (Figs. 1 and 2,

1234567

FIG.

2.

IEF

plate

as in Fig. 1 but stained

with

silver

reagent.

ISOELECTRIC

FOCUSING

ELECTROPHORESIS

PH 2.5-

6.07.0 6.0 -

I) 10.0 1234

5

FIG. 3. IEF of alkaline-soluble fraction of PGW treated with ligninolytic enzymes. (A) Samples: 1, PGW treated with Lip; 2, PGW control for LiP treatment; 3, PGW treated with MnP; 4, PGW control for MnP treatment; 5, Kraft lignin. Plate viewed under uv. (B) IEF plate as in (A) but stained with silver reagent. The pH gradient is indicated on the left. The thick arrowhead indicates sample application.

lanes l-3), and PGW (Fig. 3, lanes 2 and 4) all gave characteristic separation patterns. The bands were located in the pH range 3.5-7.0 when viewed under uv and 4.5-8.0 when stained with silver reagent. MWL was not clearly seen under uv (Fig. 1, lane 2) and lignosulfonates were not stained with silver reagent (Fig. 2, lane 3). The uv absorption of lignin depends on the presence of hydroxyl, carbonyl, and bifenyl groups and a--/3 double bonds (6). These structures also affect fluorescence, which is similarly enhanced by the increased resonance energy (7). The ampholytes, being polycarboxy-polyamine compounds, could be expected to increase the fluorescence of lignin. Reactions of phenolics with both diamines and polycarboxylic acids are known to enhance the fluorescence of phenolic compounds (7). Silver staining, conventionally applied for proteins and nucleic acids, is based on the reduction of silver ions by macromolecules and the formation of complexes between silver ions and the charged groups of the macromolecules (8,9). Silver staining has also been used for the detection of reducing phenolic compounds (10). The differences revealed in the visualization of macromolecular lignins and lignin substructure models are thus based on the differences between their functional groups. The IEF method indicated a slight modification of PGW lignin after treatment with purified ligninolytic enzymes, LiP and MnP. The intensity of uv absorption decreased with MnP treatment (arrows in Fig. 3A, lane 3). Modification by LiP is seen in Fig. 3B, lane 1: the

OF

103

LIGNIN

components in the pH range 4.0-4.8 (arrows) are only weakly stained with silver reagent. The ir spectra of samples treated with LiP showed slightly increased absorbances in the areas indicative of carboxyl and carbonyl groups in the aromatic skeleton, (wave numbers 1716,1603 cm-‘; data not shown). This is consistent with the earlier knowledge that although ligninolytic enzymes modify and cleave monomeric and dimeric lignin model compounds by oxidative action (ll), their degradative power on macromolecular lignin is rather limited (12). These results show that macromolecular alkaline-soluble lignin can be fractionated by IEF like other natural macromolecules and visualized by means of uv absorption and silver staining. The fractionation of lignin appears to be only partly dependent on the electrophoretie mobility of lignin fragments; the separation pattern is also a consequence of fractionating precipitation due to the pH gradient. The method is valid for the separation and identification of lignin samples with characteristic features; e.g., Kraft lignin, lignosulfonates, MWL, and PGW lignin gave quite distinct separation patterns. Even the slight modification of lignin by ligninolytic enzymes could be detected after separation by IEF. If scaled up, electrofocusing could provide a new fractionation method for lignin and would facilitate the identification of modified lignin preparations by ir and NMR spectroscopy. ACKNOWLEDGMENTS The excellent technical assistance Suihkonen is gratefully acknowledged.

of R. Arnkil,

M. Tikka,

and R.

REFERENCES 1. Pridham, J. B. (1963) in Methods in Polyphenol Chemistry ham, J. B., Ed.), pp. 111-124, Pergamon, Oxford.

(Prid-

2. Nummi, M., Niku-Paavola, M-L., and Raunio, V. (1986) ceedings, 3rd International Conference, Biotechnology Pulp and Paper Industry, Stockholm, 1986, p. 157. 3. Bjorkman, A. (1956) Suen. Papperstidn. 59, 447-485. 4. Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, (1981) Science 2 11,1437-1438. 5. Karhunen, E., Kantelinen, A., and Niku-Paavola, M-L. Arch. Biochem. Biophys. 279, 25-31.

in Proin the

M.

H.

(1990)

6. Goldschmid, 0. (1971) in Lignins, Occurrence, Formation, Structure and Reactions (Sarkanen, K. V., and Ludwig, C. H., Eds.), pp. 241-266, Wiley-Interscience, New York. I. Udenfriend, S. (1964) Fluorescence Assay in Biology and Meditine, 3rd ed., p. 517, Academic Press, New York. 8. Morrissey, J. H. (1981) Anal. B&hem. 117,307-310. 9. Nielsen, 315.

B. L., and Brown,

L. R. (1984)

Anal.

Biochem.

141,311-

10. Stahl, E. (1967) Diinnschicht-Chromatographie, 2nd ed., p. 849, Springer-Verlag, Berlin. 11. Tien, M., and Kirk, T. K. (1984) Proc. Natl. Acad. Sci. USA 81, 2280-2284. 12. Kirk, T. K. (1987) Philos. Trans. R. Sot. London A 321.461-474.