A Method for Biotin Labeling of Biologically Active Oligogalacturonides Using a Chemically Stable Hydrazide Linkage

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

249, 10–19 (1997)

AB972165

A Method for Biotin Labeling of Biologically Active Oligogalacturonides Using a Chemically Stable Hydrazide Linkage Brent L. Ridley,1 Mark D. Spiro, John Glushka, Peter Albersheim, Alan Darvill, and Debra Mohnen Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712

Received January 13, 1997

Oligogalacturonides (oligomers of a-1,4-D-galacturonic acid) with degrees of polymerization (DP) between 8 and 16 were labeled with biotin using a rapid and simple two-reaction protocol that yields a stable oligogalacturonide derivative. In the first reaction biotinx-hydrazide was coupled to the anomeric carbon of the reducing galacturonic acid residue by a hydrazone linkage. Carbohydrate–hydrazone linkages such as these have been widely used to label a variety of biomolecules. However, we show herein that the oligogalacturonide–hydrazone linkage is hydrolyzed in water. In the second reaction the hydrazone linkage was reduced with sodium cyanoborohydride to form a stable hydrazide. The stability of hydrazide-linked oligogalacturonides was confirmed using high-performance anion-exchange chromatography (HPAEC). The biotin and uronic acid content of the HPAEC fractions was determined using quantitative colorimetric microplate assays. Electrospray mass spectrometry and 1H NMR spectroscopy were used to confirm the structure of the HPAEC-purified biotin-derivatized oligogalacturonides. Biotin-derivatized oligogalacturonides will be useful in studies of the biological functions of oligogalacturonides. q 1997 Academic Press

Widely divergent organisms, including plants, animals, fungi, and bacteria, use carbohydrates as signal molecules (known as oligosaccharins) and as structural elements (1–7). Homopolymers of a-1,4-D-linked galactosyluronic acid residues (homogalacturonans) are abundant polysaccharides in pectin, which is a promi-

nent polyanionic structural component of plant cell walls (8). Homogalacturonan is cleaved by plant and microbial enzymes such as a-1,4-endopolygalacturonase and a-1,4-endopectate lyase. During this process oligogalacturonides with the required degree of polymerization (DP)2 for biological activity may be formed (9–11). The treatment of various tissues from a divergent group of dicotyledonous plants with oligogalacturonides at concentrations as low as 0.1 mM elicits various responses such as rapid changes in ion flux at the plasma membrane, regulation of tobacco explant morphogenesis, and induction of a variety of defense responses including the accumulation of phytoalexins (2, 12, 13). In most bioassays the oligogalacturonides with maximal biological activity have DPs between 10 and 15, but smaller or larger oligomers can be the most active in certain bioassays (2, 14). In this report we describe a method to label biologically active oligogalacturonides. Carbohydrates derivatized with labels that can be detected easily at low concentrations have been used to study the biosynthesis and deposition of structural carbohydrates (15–17) and to identify putative oligosaccharin receptors (18–20). For example, high-affinity binding proteins specific for the phytoalexin-inducing hepta-b-glucoside from the fungal pathogen Phytophthora sojae have been identified in, and solubilized from, soybean plasma membrane fractions using chemically stable radiolabeled derivatives of the ligand (18, 19). In addition, a saturable binding site for the Nacetylchitooligosaccharides that induce defense-related responses has been identified in tomato and rice 2

1

To whom correspondence should be addressed. Fax: 706-5424412. E-mail: [email protected].

Abbreviations used: DP, degree of polymerization; HPAEC–PAD, high-performance anion-exchange chromatography–pulsed amperometric detection.

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0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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suspension-cultured cells using chemically stable radiolabeled derivatives of the ligands (20, 21). However, progress toward the identification, characterization, and purification of putative oligogalacturonide receptors has been slow, due at least in part to the unavailability of stably labeled oligogalacturonides (22–25). We present evidence that oligogalacturonide–biotin hydrazone derivatives are unstable in aqueous solutions but that reduction of the hydrazone yields a stable oligogalacturonide hydrazide. This paper describes a method for the stable labeling of oligogalacturonides with biotin. MATERIALS AND METHODS

Reagents Trigalacturonic acid, avidin, biotinylated alkaline phosphatase, and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). Biotin-x-hydrazide {6-[(biotinoyl)amino]caproic acid hydrazide} was purchased form Calbiochem (La Jolla, CA). Oligogalacturonides of DPs 8, 11, and 16 were produced by partial endopolygalacturonase digestion of polygalacturonic acid (Sigma), purified by selective ethanol precipitation, and size-fractionated using QSepharose anion-exchange chromatography (11). Oligogalacturonide preparations were stored at 0807C. Aqueous solutions containing oligogalacturonides were sterilized using 0.2-mm syringe filters (Whatman, Clifton, NJ). Quantitative Colorimetric Assay for Uronic Acid Uronic acid content was quantified using the method of Blumenkranz and Asboe-Hansen (26), which was modified for use in a 96-well microplate assay. This modification provides rapid and convenient analysis of many samples (27). Samples or standards (50 mL containing 0.5–10 mg of uronic acid) were placed in polypropylene RIA vials arranged in a metal 96-position RIA vial rack (Sarstedt Inc., Newton, NC). Sodium tetraborate (12.5 mM) in concentrated sulfuric acid (300 mL) was added to each assay vial using a repeating multichannel pipet with disposable plungers (Brinkman Instruments, Inc., Westbury, NY). The assay solutions were mixed for 5 min using a microplate mixer, and the reactions were heated to 1007C for 5 min on a microplate heating block (USA/Scientific, Ocala, FL). The racks were placed in a shallow recess in the microplate heating block, which was filled with water to increase the heat transfer. After heating, the reactions were cooled for about 5 min in ice water. Color reagent [5 mL of 0.15% m-hydroxybiphenyl, 0.5% (w/v) sodium hydroxide in water] was added to each vial, and the reactions were mixed until the color fully developed (about 5 min). The entire contents of each assay vial

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were transferred to a polystyrene 96-well microplate (Nunc Inc., Naperville, IL) using the repeating multichannel pipet, and the absorbance (540 nm) was measured using a spectrophotometric microplate reader (Flow Laboratories, McLean, VA). Competitive Microplate Assay for Biotin Quantification Free and derivatized biotin content was analyzed using a competitive colorimetric enzyme assay. Polystyrene 96-well microplates (Nunc) were coated overnight with avidin (15 mg/mL in 75 mM NaCl, 0.05% NaN3 , 200 mM borate, pH 8.5). The plates were washed once with wash buffer (200 mM NaCl, 20 mM Tris, pH 7.5, 0.1% bovine serum albumin) and blocked for 30 min using 50 mL of blocking buffer (200 mM NaCl, 20 mM Tris, pH 7.5, 1% bovine serum albumin, 0.05% NaN3). The plates were washed three times with wash buffer after the blocking step and after each of the subsequent biotin–avidin binding steps. Biotin-containing samples or standards (50 mL, 0.2–1 pg) in dilution buffer (200 mM NaCl, 5 mM MgCl2 , 0.1% bovine serum albumin, 0.05% NaN3 , 20 mM Tris, pH 7.5) were allowed to bind to the avidin-coated wells for 12 h at room temperature. Subsequently, biotinylated alkaline phosphatase (50 mL, 1 mg/mL) in dilution buffer was dispensed into the wells and allowed to bind to any unoccupied biotin binding sites for 12 h at room temperature. The plates were developed with alkaline phosphatase substrate [50 mL, 0.5 mM MgCl2 , 1.66 mg/mL p-nitrophenyl phosphate, 9.6% (v/v) diethanolamine, pH 9.6]. After 10 min, the reactions were stopped with 3 M NaOH (50 mL). The absorbance of the reactant (p-nitrophenyl phosphate, 405 nm) and the product (p-nitrophenol, 492 nm) was measured, and the readings were corrected for overlapping absorbance as described (28). Production of Oligogalacturonide–Biotin Hydrazones A hydrazone linkage between oligogalacturonides and biotin was formed in the first reaction of the labeling procedure. Biotin-x-hydrazide (6.9 mg) was dissolved in sodium acetate buffer (200 mL, 20 mM, pH 4.8) by heating in a boiling water bath and ultrasonicating. After the solution had cooled to about 507C, oligogalacturonides (1 mg, NH4 salt) were added. Immediately after adding the oligogalacturonides, 10% (v/v) acetic acid in methanol (1 mL) was added with vigorous vortexing. The aqueous solvent was removed by evaporation using a stream of N2 (407C, approximately 30 min). Acetic acid in methanol [1 mL, 10% (v/v)] was added repeatedly (31) to the biotinylation reaction residue, which was not completely soluble, and the mixture was briefly ultrasonicated to yield a fine suspension. After each addition the solvent was evaporated using a stream of N2 (407C, approximately 15 min). Finally,

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methanol (1 mL) was added repeatedly (41) to the reaction residue and evaporated to remove any residual acetic acid. The biotin hydrazone-linked oligogalacturonides were stored dry at 0807C or reduced immediately (see below). Reduction of Hydrazones to Hydrazides The unstable hydrazone linkage between biotin and the oligogalacturonides was reduced to form a stable hydrazide linkage (29). The reduction reaction was assembled as quickly as possible to avoid unwanted side reactions (e.g., osazone formation) (30, 31). The dry biotinylation reaction residue was rapidly suspended in 180 mL of water using an ultrasonicator, and NaCNBH3 was added immediately (20 mL, 230 mg/mL). Hydrochloric acid (16 mL, 1 M) was added in 2-mL aliquots at 10-s intervals with mixing. After HCl addition, the reaction residue was fully dissolved, and the pH of the sample was about 3.2. The reaction was incubated for 30 min at room temperature with constant stirring. The reaction products were purified by one of two methods depending on the DP of the oligogalacturonides derivatized. When the derivatized oligogalacturonides were DP 8 or larger, the biotinylation reaction mixture was precipitated with 1 M NH4HCO2 in ethanol (3 mL, 0207C, Ç18 h) and the precipitate was collected by centrifugation (5000g). The pellet was repeatedly washed (31) in ethanol (1 mL) and collected by centrifugation (5000g). The final pellet, which was dried under a stream of N2 and stored at 0807C, contained a mixture of biotin-xhydrazide-linked and underivatized oligogalacturonides with the NaCNBH3 and most of the unreacted biotin-x-hydrazide removed. Biotin-x-hydrazide-derivatized and underivatized oligogalacturonides of DP £ 7 do not precipitate under the conditions described above. Thus, the unreacted NaCNBH3 was removed from the trigalacturonide biotinylation reaction mixtures by extensive dialysis against aqueous 1% acetic acid using 500 MWCO dialysis tubing (Spectrum Medical Industries Inc., Los Angeles, CA). This method does not remove the unreacted biotin-x-hydrazide. However, free biotin hydrazide does not interfere with subsequent chromatographic analysis of the biotinylated oligogalacturonides. Analysis of Biotinylated Oligogalacturonides Using HPAEC–PAD Biotinylated oligogalacturonides were analyzed by ion-exchange chromatography on a Carbopac PA1 or a Carbopac PA100 column depending on their DP. The analysis was conducted using a Dionex metal-free HPLC pump (Model AGP) with a pulsed amperometric detector (PAD) at a flow rate of 1 mL/min (Dionex, Sunnyvale, CA). Prior to their use, all eluants were

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filtered using a 0.2-mm nylon filter and degassed by helium sparging. The PAD detector had a gold-working electrode and was operated as described (11). Sodium hydroxide was added (0.4 M, 0.33 mL/min) to the column eluate prior to PAD detection, since high pH increases the sensitivity of carbohydrate detection. Trigalacturonide biotinylation reaction mixtures (10–20 mg of galactosyluronic acids as determined colorimetrically) were separated on a Carbopac PA1 column (Dionex) using linear sodium acetate gradients (5–350 mM over 75 min or 150–300 mM over 60 min). Biotinylation reaction mixtures containing larger oligomers (20–50 mg of galactosyluronic acid) were separated on a Carbopac PA100 column using linear sodium acetate gradients (for example, 430–540 mM over 40 min for the undecamer, 460–560 mM over 40 min for the tridecamer) or potassium oxalate gradients (for example, 85–135 mM over 40 min for the undecamer, 120–170 mM over 40 min for the tridecamer). Purification of Biotinylated Oligogalacturonides Using HPAEC–PAD Biotin-labeled oligogalacturonides having DPs of 8 or greater were purified using a Carbopac PA100 column and PAD as described. Portions of biotinylation reaction mixtures (Ç200 mg of galactosyluronic acid) were separated using sodium acetate gradients (for example, 480–520 mM over 20 min for the undecamer, 500–540 mM over 20 min for the tridecamer). The use of shallow gradients and short run times resulted in improved separation of the reaction components and quicker purification. The performance of the columns gradually deteriorated with use. Therefore, the columns were periodically regenerated with sequential washes of HCl (1 M, 1 mL/min, 1 h), water (1 mL/min, 15 min), and NaOH (3 M, 1 mL/min, 1 h). To increase the efficiency of carbohydrate detection, sodium hydroxide is normally mixed (0.4 M, 0.33 mL/ min) with the column eluate before it enters the PAD. However, high pH destroys oligogalacturonide–biotin hydrazides. Consequently, NaOH could not be added to the column eluate while oligogalacturonide–biotin hydrazide-containing fractions were being collected during purification. Therefore, pilot runs were used to determine the retention times of the desired fractions. During purification runs, PAD was used to locate the beginning of the oligogalacturonide–biotin hydrazidecontaining peak. When the desired peak began to elute, a three-way valve (Rainin, Ridgefield, NJ) in the tubing between the column and the PAD was switched, diverting the eluate prior to NaOH addition. Simultaneously, the detector was turned off and the oligogalacturonide– biotin hydrazide-containing fraction was collected. The oligogalacturonide–biotin hydrazide-containing fractions were precipitated with ethanol (six volumes,

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0207C, Ç18 h) and the precipitate was collected by centrifugation (5000g). The pellet was repeatedly washed (31) in ethanol (1 mL) and collected by centrifugation (5000g). The final pellet was dried under a stream of N2 . Mass Spectral Analysis of Biotinylated Oligogalacturonides Samples of purified undecagalacturonide–biotin hydrazide and tridecagalacturonide–biotin hydrazide were analyzed using electrospray mass spectrometry on an API III biomolecular mass analyzer (PE-Sciex, Thornhill, Canada). The samples were analyzed using the positive-ion mode with an ion spray potential of 5000 V and an orifice potential of 35 V. HPAEC-purified biotinylated oligogalacturonides (1 mg/mL in aq 2 mM NH4HCO2) were introduced into the electrospray source at 2 mL per minute using a Harvard 22 syringe infusion pump. The mass range between 1100 and 1500 amu was scanned, and 10 scans were collected and averaged. Reaction mixtures containing trigalacturonide–biotin hydrazide were analyzed by fast atom bombardment mass spectrometry (FAB–MS) using a VG ZABSE mass spectrometer (VG Analytical, Altringham, UK) as described (11). Samples of reaction mixtures (1 ml, 10 mg/mL) were mixed with thioglycerol (1 mL) and HCl (0.5 mL) on the mass spectrometer probe tip just prior to the analysis. The mass spectrometer was operated in the negative-ion mode. NMR spectral analysis of biotinylated oligogalacturonides. Solutions (1 mg/mL) of HPAEC-purified biotinylated tridecagalacturonide were repeatedly exchanged in D2O and dissolved in 99.96% D2O in preparation for NMR analysis. Proton spectra were collected on a Bruker AMX 600-MHz spectrometer at 257C. The signal from residual HDO was reduced by irradiation with a low-power presaturation pulse. A 2D TOCSY dataset was collected using a 50-ms DIPSI mixing sequence [data not shown (32 – 34)]. Data were processed using Felix software (Molecular Simulations Inc., San Diego, CA). RESULTS

Scheme for Biotinylation of Oligogalacturonides The two-reaction procedure depicted in Fig. 1 was used to derivatize several sizes of biologically active oligogalacturonides with biotin by formation of a stable hydrazide linkage. First, the aldehyde function of the reducing terminal galactosyluronic acid residue is coupled to the acid hydrazide function of biotin-x-hydrazide via a hydrazone linkage (Fig. 1, Reaction 1). The coupling reaction is driven toward product formation by repeatedly adding and evaporating 10% acetic acid

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in methanol, thereby removing water, a reaction product. Next, the hydrazone linkage was reduced to form a stable hydrazide (Fig. 1, Reaction 2). Instability of Biotin Hydrazones of Oligogalacturonides A review of literature on the hydrazones of sugars suggested that the oligogalacturonide–hydrazone linkages might be unstable (30, 31). Since it is commercially available, we used trigalacturonide to determine the stability of the oligogalacturonide–hydrazone linkages in water and to establish reaction conditions for efficient hydrazone formation. The trigalacturonide (Fig. 2A) was coupled to biotin-x-hydrazide without reduction. The product was dissolved in water and immediately analyzed by HPAEC–PAD (Fig. 2B). The peak corresponding to the trigalacturonide–biotin hydrazone (Fig. 2B, peak 1) accounted for about 93% of the total trigalacturonide in the biotinylation reaction mixture as determined by PAD, while the peak corresponding to free trigalacturonide (Fig. 2B, peak 2) accounted for only about 7% of the total trigalacturonide. Colorimetric assays established that the trigalacturonide– biotin hydrazone peak (Fig. 2B, peak 1) contained equimolar amounts of biotin and trigalacturonide. The galacturonic acid content determined by quantitative colorimetric assays was found to be proportional to the PAD response (peak area) for both biotin-derivatized and underivatized trigalacturonide. The trigalacturonide was used to determine the stability of oligogalacturonide–hydrazones in water. The remainder of the reaction mixture shown in Fig. 2B was incubated 12 h in water at room temperature and then analyzed by HPAEC–PAD (Fig. 2C). The amount of free trigalacturonide in the reaction mixture increased from 7 to 36% of the PAD-detectable material (Fig. 2C, peak 2), demonstrating that hydrazone-linked oligogalacturonides are unstable in water. After the analysis shown in Fig. 2C, the remaining reaction mixture was again subjected to the coupling reaction of the labeling procedure and analyzed by HPAEC–PAD (Fig. 2D). Almost all of the free trigalacturonide was again converted to the trigalacturonide–biotin hydrazone (Fig. 2D, peak 1), demonstrating that the hydrazone linkage can be repeatedly formed and hydrolyzed. The structure of the trigalacturonide–biotin hydrazone would have been difficult to confirm using mass spectrometry or NMR spectroscopy because the hydrazone linkage is unstable in water. Therefore, we confirmed the structure of the trigalacturonide–biotin hydrazone through structural characterization of the trigalacturonide–biotin hydrazide derived from it. A trigalacturonide biotinylation reaction mixture was coupled (Fig. 1, Reaction 1), reduced (Fig. 1, Reaction 2), dialyzed, and then analyzed by HPAEC–PAD using

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FIG. 1. The two-step procedure used to biotinylate oligogalacturonides. (1) Biotin hydrazide is coupled to an oligogalacturonide through a hydrazone linkage. (2) The oligogalacturonide–biotin hydrazone is reduced to an oligogalacturonide–biotin hydrazide linkage.

a Carbopac PA 1 column as described. The predominant peak observed (Ç91% of the PAD-detectable material) had a different retention time than the hydrazone (data not shown). As expected for the trigalacturonide–biotin hydrazide, prolonged incubation of the sample in water did not affect the amount of PAD-detectable material in this peak. The dialyzed trigalacturonide–biotin hydrazide reaction mixture was analyzed by FAB–MS in the negative-ion mode. The largest two peaks in the mass spectrum were at m/z 900.8 and 882.7. These peaks corresponded to the trigalacturonide–biotin hydrazide (calculated m/z [M 0 H]//1 Å 900.3) and the lactone form of the trigalacturonide–biotin hydrazide (calculated m/z [M 0 H]//1 Å 882.7). The lactone of trigalacturonide–biotin hydrazide, which is an internal ester formed between C2 and C6 of the modified reducing terminal galacturonic acid residue, is known to form at low pH and was undoubtedly formed in this sample when the pH of the sample was lowered in preparation for FAB–MS analysis (11, 35). Labeling of Biologically Active Oligogalacturonides with Biotin Hydrazide The yield of tridecagalacturonide–biotin hydrazone after the coupling reaction (Fig. 1, Reaction 1) was deter-

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mined using HPAEC–PAD. Tridecagalacturonide (Fig. 3A) was coupled to biotin-x-hydrazide and then analyzed immediately after having been dissolved in water (Fig. 3B). The peak corresponding to the tridecagalacturonide–biotin hydrazone (Fig. 3B, peak 3), which contains equimolar amounts of tridecagalacturonide and biotin (determined colorimetrically), typically accounts for 60– 80% of the tridecagalacturonide in the reaction mixture. The reaction product also contained significant amounts of free biotin-x-hydrazide (Fig. 3B, peak 1) and free tridecagalacturonide (Fig. 3B, peak 2; compare to Fig. 3A). The PAD response (peak area) to biotin-derivatized and underivatized tridecagalacturonide was found to be proportional to the galacturonic acid content determined by the quantitative colorimetric assays. The tridecagalacturonide–biotin hydrazone is rapidly hydrolyzed in aqueous solution, releasing free tridecagalacturonide and biotin-x-hydrazide (data not shown). Since the tridecagalacturonide–biotin hydrazone is unstable in water, it could not readily be obtained in pure form and was not studied further. However, the tridecagalacturonide–biotin hydrazone is stable in dry biotinylation reaction mixtures when stored at 0807C. The tridecagalacturonide–biotin hydrazone was reduced (Fig. 1, Reaction 2) to form a stable biotin hydraz-

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Oligogalacturonides with DPs of 3, 8, 10, 11, 13, and 16 were labeled with biotin via a hydrazide linkage and fractionated by HPAEC–PAD. The yield of biotinylated oligogalacturonide, as determined by HPAEC–PAD, decreased as the DP of the oligogalacturonide being derivatized was increased. Accordingly, the yield of oligogalacturonide–biotin hydrazide from a single reaction using DP 8 was 88%, using DP 10 was 83%, using DP 11 was 76%, and using DP 16 was 39%. Furthermore, the average yield of trigalacturonide–biotin hydrazide from six reactions was 93%, whereas the average yield of tridecagalacturonide–biotin hydrazide from four reactions was 72%. The efficiency of the reduction reaction approaches 100%, since the overall yield of the oligogalacturonide–biotin hydrazides after the reduction reaction (Fig. 1, Reaction 2) is always about the same as the yield of oligogalacturonide–biotin hydrazones after the coupling reaction, regardless of the DP of the oligogalacturonide. Therefore, it is likely that the lower yields obtained when longer oligo-

FIG. 2. HPAEC–PAD fractionation of a trigalacturonide biotinylation reaction mixture after the coupling reaction (Fig. 1, Reaction 1) using a linear sodium acetate gradient (5–350 mM over 75 min) on a Carbopac PA1 column. (A) Chromatography of the trigalacturonide. (B) A trigalacturonide biotinylation reaction without NaCNBH3 reduction. The reaction mixture was dissolved in water and analyzed immediately. Peak 1 is the trigalacturonide–biotin hydrazone. (C) The reaction mixture used in B was incubated 12 h in water at room temperature and chromatographed. (D) The reaction mixture used in C was subjected to the coupling reaction. After the coupling reaction, the reaction mixture was dissolved in water and analyzed immediately. The peak shape distortion in D was caused by overloading the column.

ide. Typically, the yield of tridecagalacturonide–biotin hydrazide, as determined by PAD, was 60–80% of the total tridecagalacturonide in the reaction mixture. The separation of a reduced tridecagalacturonide biotinylation reaction mixture by HPAEC – PAD is presented in Fig. 3C. Ethanol precipitation has removed the NaCNBH3 and most of the free biotin-x-hydrazide (Fig. 3C, peak 1; compare to Fig. 3B, peak 1). The peak corresponding to the tridecagalacturonide–biotin hydrazide (Fig. 3C, peak 4) has equimolar amounts of tridecagalacturonide and biotin (determined colorimetrically). The reduced biotinylation reaction mixture also contained significant 15–20% free tridecagalacturonide (Fig. 3C, peak 2; compare to Fig. 3A) but no detectable tridecagalacturonide–biotin hydrazone (compare Fig. 3C to Fig. 3B, peak 3). Tridecagalacturonide–biotin hydrazide is stable indefinitely when stored in sterile aqueous solutions at neutral pH.

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FIG. 3. HPAEC–PAD separation of a tridecagalacturonide biotinylation reaction mixture using a linear potassium oxalate gradient (120–170 mM over 40 min) on a Carbopac PA100 column. (A) Chromatography of the Q-Sepharose purified tridecagalacturonide used in this experiment. Peak 2 contains the tridecagalacturonide. (B) Chromatography of a tridecagalacturonide biotinylation reaction mixture after the coupling reaction but prior to NaCNBH3 reduction. Peak 1 contains free biotin-x-hydrazide, and peak 3 contains the tridecagalacturonide–biotin hydrazone. (C) Chromatography of a tridecagalacturonide biotinylation reaction mixture after the reduction reaction. Peak 4 contains the tridecagalacturonide–biotin hydrazide.

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galacturonides are reacted with biotin-x-hydrazide result from reduced efficiency in the coupling reaction (Fig. 1, Reaction 1). Purification of Biotinylated Oligogalacturonides Biotin hydrazides of oligogalacturonides with DPs of 8, 10, 11, 13, and 16 were purified on a Carbopac PA100 using sodium acetate gradients and analyzed for purity on the same column using potassium oxalate gradients. The analysis using potassium oxalate gradients provided independent verification of the purity, since the reaction components, including free oligogalacturonides and their hydrazone or hydrazide derivatives, elute in a different order when potassium oxalate rather than sodium acetate is used in the eluate. The recovery of the oligogalacturonide–biotin hydrazides following the HPAEC purification step typically was about 75–80%. Some of the biotinylated oligogalacturonides lost during purification were retained on the column, resulting in a gradual deterioration of its performance. Periodic cleaning with NaOH and HCl was required to restore the column’s performance. Chemical Characterization of a Biotinylated Tridecagalacturonide The structure of the HPAEC-purified tridecagalacturonide–biotin hydrazide was confirmed using electrospray mass spectrometry and 1H NMR spectroscopy. The positive-ion electrospray mass spectrum of the biotinylated tridecagalacturonide gave a series of doubly charged ions with sodium, ammonium, and potassium adducts that is consistent with a single biotin-x-hydrazide of the tridecagalacturonide (Fig. 4). For example, the peak at m/z 1332.5 corresponds to the doubly charged pseudomolecular ion of tridecagalacturonide– biotin hydrazide, the peak at m/z 1343.5 corresponds to the doubly charged pseudomolecular ion of tridecagalacturonide–biotin hydrazide with a sodium adduct, and the peak at m/z 1354.5 corresponds to the doubly charged pseudomolecular ion of tridecagalacturonide– biotin hydrazide with two sodium adducts. The 1H NMR spectra from 1D and 2D experiments with HPAEC-purified tridecagalacturonide–biotin hydrazide are completely consistent with the structure proposed. The five most intense signals (Table 1 and Fig. 5, peaks ga1–ga5), which result from the ring protons of the internal repeating galacturonosyl residues, have the expected chemical shifts (36). As expected, the signals from the protons of the nonreducing terminal galacturonic acid residue (Table 1) have chemical shifts different from the internal residues (36). The signals due to the biotin moiety have chemical shifts (Fig. 5, all peaks labeled with a ‘‘b’’) that match the values reported (37). After integration, the ratio of signals arising from the biotin hydrazide to signals arising

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FIG. 4. Electrospray mass spectrum of the biotin-x-hydrazide of the tridecagalacturonide. A solution of HPAEC-purified, biotin-labeled tridecagalacturonide (1 mg/mL) in aq 2 mM ammonium formate was infused into the electrospray source at 2 mL/min. The peaks are labeled with their observed m/z values. The calculated m/z values for doubly charged tridecagalacturonide–biotin hydrazide pseudomolecular ions with ammonium, sodium, and potassium adducts corresponding to the peaks observed are as follows: ([M / 2H]/)/2, 1332.7; ([M / NH4 / H]2/)/2, 1341.2; ([M / Na / H]2/)/2, 1343.7; ([M / 2NH4]2/)/2, 1349.7; ([M / K / H]2/)/2, 1351.7; ([M / 2Na]2/)/2, 1354.7; ([M / NH4 / 2Na 0 H]2/)/2, 1362.7; and ([M / 3Na/ 0 H]2/)/ 2, 1365.2.

from the carbohydrate shows that there is one biotin group per tridecamer. The absence of signals not attributed to the tridecagalacturonide–biotin hydrazide confirmed that the preparation was pure. A 2D TOCSY spectrum of the tridecagalacturonide– biotin hydrazide was used to assign the signals of the modified galactosyluronic acid residue at the former reducing end, the galactosyluronic acid residue adjacent to it, and the biotin-x-hydrazide group (data not shown). The signals from the galactosyluronic acid residue adjacent to the residue linked to biotin-x-hydrazide (formerly the reducing end residue) have chemical shifts that are different from those expected for unmodified tridecagalacturonide (Table 1). The former reducing end galacturonic acid residue is clearly no longer a pyranose. There are two protons on carbon 1 instead of one (Fig. 5, 1*) and their signals are shifted upfield to d 2.95 (Table 1). This is consistent with the presence of a methylene amine function at carbon 1, which is the expected structure if carbon 1 is derivatized with a hydrazide (Fig. 1). The remaining signals belonging to the former reducing galacturonic acid residue also have chemical shifts that differ from those of ordinary oligogalacturonides, demonstrating that this residue

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BIOTIN LABELING OF OLIGOGALACTURONIDES TABLE 1

Proton Chemical Shifts in D2O at 257C of HPAEC-Purified Tridecagalacturonide–Biotin Hydrazide Galacturonosyl residue

H1

H2

H3

H4

H5

Hydrazide-linked Adjacent to hydrazide-linked Internal Nonreducing terminal

2.95a 5.14 5.06 5.05

3.90 3.96 3.76 3.91

3.68 3.82 4.00 3.70

4.23 4.41 4.41 4.25

4.30 4.76 4.76 4.76

Biotin ringsb 2 3 4 5

Biotin alkyl chainb 3.34 4.43 4.61 3.00, 2.78

a b g d

2.25 1.60 1.40 1.6, 1.71

Linker chainb

e h z

3.17 1.3, 1.51 2.23

Note. All values are expressed in ppm, relative to external TSP. The appropriate connectivities were determined by a 2D TOCSY spectrum (data not shown). The chemical shifts of the peaks due to the biotin moiety (Fig. 5, peaks b) match literature values (37). The nomenclature for the protons of the biotin rings, the alkyl chain of biotin, and the linker chain is shown in Fig. 5. The signals due to the alkyl linker chain are found in the same region of the spectrum as the biotin alkyl chain, except for the methylene resonance at d3.17 (Table 1, linker chain e). Integration of the signals from the linker chain and the former reducing and residue indicates that there is one biotin hydrazide group per oligomer. a All values are expressed in ppm relative to external TSP. b The nomenclature for the protons of the biotin rings, the alkyl chain of biotin, and the linker chain is shown in Fig. 5.

has been chemically modified (Table 1). This is most pronounced with the proton at carbon 5 which has a chemical shift of d 4.30 instead of the expected d 4.76. In

addition, the characteristic vicinal coupling constants found in the galactopyranose configuration are not present. For example, 3J(H2-H3) is less than 1 Hz compared to 10 Hz when in the galactopyranose configuration, and 3J(H3-H4) is 8 Hz compared to less than 1 Hz found in the galactopyranose configuration. DISCUSSION

FIG. 5. The 600-MHz NMR spectrum of the biotinylated tridecagalacturonide. The diagram gives the nomenclature for the protons of the biotin-x-hydrazide. The peaks labeled ‘‘1*–5*’’ arise from the former reducing end residue, and those labeled ‘‘b’’ arise from the biotin hydrazide. The intense signals labeled ‘‘ga1–ga5’’ arise from 10 of the internal galacturonosyl residues. Low intensity signals that are not labeled arise from the galacturonosyl residue at the nonreducing terminal galactosyluronic acid residue and the residue adjacent to the former reducing terminal galacturonic acid residue.

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A simple two-reaction procedure for efficient derivatization of oligogalacturonides with biotin-x-hydrazide has been developed. The carbonyl function of the reducing galacturonosyl residue is first coupled to biotin-x-hydrazide via a hydrazone linkage. The formation of sugar hydrazones is most efficient when the reaction medium is an organic solvent such as methanol (30, 38). However, oligogalacturonides larger than DP 4 are essentially insoluble in methanol. To alleviate this potential problem, repeated suspension in 10% acetic acid in methanol and drying of the oligogalacturonide biotinylation reaction mixture were used to drive the reaction by removing the reaction product, water. This coupling method is useful for oligogalacturonides of DP 8 – 16 despite the fact that the reactants are not completely soluble. However, the yields are lower when derivatizing oligogalacturonides with higher DPs. This effect is probably due to increased self-aggregation and decreased solubility of the longer oligogalacturonides (39). The coupling method we have described is expected to be more efficient with neutral sugars than with hexosyluronic acids. Neutral sugars are more soluble in organic solvents than uronic acids. In addition, the

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RIDLEY ET AL.

formation of sugar hydrazones is slower with hexuronic acids than with neutral sugars because the carbon six carboxylate of hexuronic acids stabilizes the hemiacetal ring, thus making the reducing carbonyl less available to react with a hydrazide reagent (30). Therefore, it is likely that the biotinylation procedure described herein can be adapted for derivatization of other carbohydrates with hydrazides, including biotin-x-hydrazide. The hydrazone linkage between biotin and the carbohydrate is reduced to form a hydrazide linkage. The reduction reaction approaches 100% efficiency regardless of the DP of the oligogalacturonide being biotinylated. The reduction is necessary because hydrazone– carbohydrate linkages are hydrolyzed in aqueous solutions (30, 31, 40, 41). Despite the possibility of hydrolysis of carbohydrate–hydrazones, numerous investigators have used glycoproteins (see, for example, 42–44) or carbohydrates (see, for example, 24, 25, 45, 46) labeled via hydrazone linkages. Reduction of carbohydrate–hydrazone linkages to form stable hydrazide linkages provides a means to obtain labeled molecules with greater stability in aqueous environments. Stable biotin-labeled oligogalacturonides provide attractive alternatives to radiolabeled oligogalacturonides since they are safe to use, are detectable in sensitive assays, and can be stored indefinitely (35). Biotinlabeled oligogalacturonides are easily detectable using cytochemical techniques and have been used to investigate cellular recognition, uptake, and transport of oligogalacturonides (24, 25, 45). Biotinylated oligogalacturonides have also be used as exogenous acceptors to study the biosynthesis of homogalacturonan (47, 48, unpublished results). Biotinylated oligogalacturonides are useful in studies of oligogalacturonide signaling provided that the reducing end modification does not destroy the biological activity. Recently it has been demonstrated that these reducing end derivatized oligogalacturonides retain most or all of their biological activity in some plant bioassays (unpublished results). Thus, biotinylated oligogalacturonides could be used in studies designed to identify and purify oligogalacturonide receptors and to study signal transduction pathways mediating at least some of the biological activities of oligogalacturonides (46). A particular advantage of biotin-derivatized signal molecules is that they can be bound to avidin-coupled agarose for use in affinity purification of receptor proteins (49). Reymond et al. have recently reported using NaCNBH3 reduced biotin-x-hydrazide-labeled oligogalacturonides to study an oligogalacturonide binding protein (50). The structure of the biotinylated oligogalacturonides was not chemically characterized and the yield of the reaction was not reported. However, the work reported here suggests that the procedure used by Reymond et al. couples biotin-x-

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hydrazide to oligogalacturonides via a stable hydrazide linkage. ACKNOWLEDGMENTS We thank V. Puvanesarajah and M. O’Neill for performing the mass spectral analysis and for many helpful discussions. We thank C. Bergmann for technical advice. This research is supported by U.S. Department of Energy (DOE) Grant DE-FG02-96ER20221 and by the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates.

REFERENCES 1. McCann, M. C., and Roberts, K. (1991) in Cytoskeletal Basis of Plant Growth and Form (Lloyd, C. W., Ed.), pp. 109–129, Academic Press, London. 2. Coˆte´, F., and Hahn, M. G. (1994) Plant Mol. Biol. 26, 1379–1411. 3. Satiat-Jeunemaitre, B. (1992) Tissue Cell 24, 315–334. 4. Varki, A. (1993) Glycobiology 3, 97–130. 5. Boller, T. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 189–214. 6. Ghuysen, J.-M., and Hackenbeck, R. (Eds.) (1994) The Bacterial Cell Wall, Elsevier, Amsterdam. 7. Wessels, J. G. H. (1993) New Phytol. 123, 397–413. 8. O’Neill, M., Albersheim, P., and Darvill, A. (1990) in Methods in Plant Biochemistry (Dey, P. M., Ed.), pp. 415–441, Academic Press, London. 9. Bergmann, C. W., Ito, Y., Singer, D., Albersheim, P., Darvill, A. G., Benhamou, N., Nuss, L., Salvi, G., Cervone, F., and De Lorenzo, G. (1994) Plant J. 5, 625–634. 10. Melotto, E., Greve, L. C., and Labavitch, J. M. (1994) Plant Physiol. 106, 575–581. 11. Spiro, M. D., Kates, K. A., Koller, A. L., O’Neill, M. A., Albersheim, P., and Darvill, A. G. (1993) Carbohydr. Res. 247, 9–20. 12. Mathieu, Y., Kurkdjian, A., Xia, H., Guern, J., Koller, A., Spiro, M. D., O’Neill, M., Albersheim, P., and Darvill, A. (1991) Plant J. 1, 333–343. 13. Marfa`, V., Gollin, D. J., Eberhard, S., Mohnen, D., Darvill, A., and Albersheim, P. (1991) Plant J. 1, 217–225. 14. Weber, J., Olsen, O., Wegener, C., and von Wettstein, D. (1996) Physiol. Mol. Plant Pathol. 48, 389–401. 15. Price, N. P. J., Kelly, T. M., Raetz, C. R. H., and Carlson, R. W. (1994) J. Bacteriol. 176, 4646–4655. 16. Smith, R. C., and Fry, S. C. (1991) Biochem. J. 279, 529–535. 17. D’Souza, C., Sharma, C. B., and Elbein, A. D. (1992) Anal. Biochem. 203,211–217. 18. Cheong, J.-J., Alba, R., Coˆte´, F., Enkerli, J., and Hahn, M. G. (1993) Plant Physiol. 103, 1173–1182. 19. Frey, T., Cosio, E. G., and Ebel, J. (1993) Phytochemistry 32, 543–550. 20. Baureithel, K., Felix, G., and Boller, T. (1994) J. Biol. Chem. 269, 17931–17938. 21. Shibuya, N., Kaku, H., Kuchitsu, K., and Maliarik, M. J. (1993) FEBS Lett. 329, 75–78. 22. Jacinto, T., Farmer, E. E., and Ryan, C. A. (1993) Plant Physiol. 103, 1393–1397. 23. Farmer, E. E., Kunz, B., Paul-Peltzer, K., Grunberger, S., Weber, J., Meier, S., and Caldelari, D. (1996) 8th International Congress, Molecular Plant–Microbe Interactions, Knoxville, TN, July 14– 19 (Abstract).

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BIOTIN LABELING OF OLIGOGALACTURONIDES 24. Horn, M. A., Heinstein, P. F., and Low, P. S. (1989) Plant Cell 1, 1003–1009. 25. Diekmann, W., Herkt, B., Low, P. S., Nu¨rnberger, T., Scheel, D., Terschu¨ren, C., and Robinson, D. G. (1994) Planta 195, 126– 137. 26. Blumenkrantz, N. J., and Asboe-Hansen, B. (1973) Anal. Biochem. 54, 484–489. 27. Wicker, L., and Leiting, V. A. (1995) Anal. Biochem. 229, 148– 150. 28. Goding, J. W. (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, San Diego. 29. Lane, C. F. (1975) Synthesis 3, 135–146. 30. Mester, L., and El Khadem, H. S. (1980) in The Carbohydrates, Vol. IB, Chemistry and Biochemistry (Pigman, W., Horton, D., and Wander, J. D., Eds.), pp. 929–988, Academic Press, New York. 31. Percival, E. G. (1948) in Advances in Carbohydrate Chemistry (Pigman, W. W., and Wolfefrom, M. L., Eds.), Academic Press, New York. 32. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521–528. 33. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355–360. 34. Rucker, S. P., and Shaka, A. J. (1989) Mol. Phys. 68, 509–517. 35. Spiro, M. D., Ridley, B. L., Glushka, J., Darvill, A., and Albersheim, P. (1996) Carbohydr. Res. 290, 147–157.

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36. Low, V.-M., Hahn, M. G., and van Hahlbeek, H. V. (1994) Carbohydr. Res. 255, 271–284. 37. Ikura, M., and Hikichi, K., (1982) Org. Mag. Reson. 20, 266– 273. 38. Helferich, B., and Schirp, H. (1951) Chem. Ber. 84, 469–471. 39. Kohn, R. (1975) Pure Appl. Chem. 42, 371–397 40. King, T. P., Zhao, S. W., and Lamb, T. (1986) Biochemistry 25, 5774–5779. 41. Kondejewski, L. H., Kralovec, J. A., Blair, A. H., and Ghose, T. (1994) Bioconjugate Chem. 5, 602–611. 42. O’Shannessy, D. J., Dobersen, M. J., and Quarles, R. H. (1984) Immunol. Lett., 273–277. 43. O’Shannessy, D. J., Voorstad, P. J., and Quarles, R. H. (1987) Anal. Biochem. 163, 204–209. 44. Bayer, E. A., Ben-Hur, H., and Wilchek, M. (1988) Anal. Biochem. 170, 271–281. 45. Low, P. S., Legendre, L., Heinstein, P. F., and Horn, M. A. (1993) J. Exp. Bot. 44(Suppl.) 269–274. 46. Shinohara, Y., Sota, H., Gotoh, M., Hasebe, M., Tosu, M., Nakao, J., and Hasegawa, Y. (1996) Anal. Chem. 68, 2573–2579. 47. Doong, R. L., Liljebjelke, K., Fralish, G., Kumar, A., and Mohnen, D. (1995) Plant Physiol. 111, 141–152. 48. Doong, R. L., Smith, J. J., and Mohnen, D. (1996) Plant Physiol. 111s, 101. 49. Green, M. N., and Toms, E. J. (1973) Biochem. J. 133, 687–700. 50. Reymond, P., Kunz, B., Paul-Pletzer, K., Grimm, R., Eckerskorn, C., and Farmer, E. E. (1996) Plant Cell 8, 2265.

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