Barley cultivar dixrimination by glycoprotein blotting

323

Original papers Walter Weiss Wilhelm Postel Angelika Gorg Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Technische Universitat Miinchen, Freising-Weihenstephan

Barley cultivar discrimination: I. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and glycoprotein blotting Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcoholsoluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glywprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviouslywere not.As a whole,glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.

1 Introduction

they could not be discriminated by their protein patterns, stained with Coomassie Brilliant Blue.

The correct and reliable identification of barley (Hordeum vulgare L.) cultivars is of great importance for the malting and brewing industries, as well as for plant breeders, because malting and brewing properties and the resistance to certain fungal or viral diseases are cultivar-dependent. Nowadays, barley cultivar discrimination is most often performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or acid PAGE ofthe alcohol soluble endosperm proteins, the hordeins [ 11. However, a number of cultivars remain indistinguishable by hordein electrophoresis because most European barley cultivars are closely related. This narrow genetic background and, additionally, a genetic linkage of the hordein loci (cJ: [2]) results in only a limited number of different hordein patterns as exhibited by one-dimensional gel electrophoresis. Better discrimination of closely related cultivars may be achieved either by applying electrophoretic methods with improved resolution, such as isoelectric focusing and two-dimensional electrophoresis, or by analyzing not only the protein fraction stained with Coomassie Brilliant Blue or silver but also other protein systems, detected by specific stains such as glycoproteins or enzymes. With respect to glycoproteins, Shah and Stegemann [3] succeeded in differentiating two wheat cultivars by their glycoprotein patterns although

Since 1969 [4],a great number of methods for detecting glycoproteins in polyacrylamide or agarose gels have been reported [4-91 (see also Table 1). However, all of these methods suffer from several disadvantages, such as low resolution, low sensitivity, high background staining, or time-consuming protocols; additionally, they do not work well in sodium dodecyl sulfate-containing gels. A major breakthrough was achieved by the introduction of an electrophoretic transfer method of electrophoretically separated proteins from polyacrylamide gels onto immobilizing membranes [lo]. These immobilized proteins cannot diffuse or be washed out, they are readily accessible to reagents or high-molecular ligands such as lectins, and incubation or washing steps can be performed easily. There are two major methods, although basically different from each other, to detect glycoproteins bound to immobilizing membranes (see Table 1): (i) using labeled chemicals that react with aldehyde groups formed by periodate oxidation of vicinal hydroxyls of the carbohydrate moieties of glycoproteins [ll], and (ii), the highly specific detection of certain sugar residues by means of lectins (i.e. carbohydrate binding proteins), followed by a visualization step [12-161.

Correspondence: Priv. Doz. Dr. A. Gorg, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Technische Universitlt Miinchen, DW-8050 Freising-Weihenstephan, Germany Abbreviations: BSA, bovine serum albumin; Con A, concanavalin A; DAB, 3,3’ diaminobenzidine; HRP, horseradish peroxidase; M , relative molecular mass; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; PVP, polyvinylpyrrolidone; SDS, sodium dodecyl sulfate; TBS, Tris buffered saline; Tris, tris(hydroxymethy1)aminomethane

C)VCH Verlagsgesellschaft m b H , D-6940Weinheim, 1991

In this study we describe two fast, inexpensive, and easy-topefform detection methods for blotted glycoproteins after SDS-PAGE, whose suitability for routine use in barley cultivar discrimination was tested. These two methods are the detection of periodate oxidized glycoproteins with the help of dansylhydrazine, and the detection of glucose- or mannose-containing glycoproteins by the concanavalin A/peroxidase (Con A/HRP) method [14, 151. The usefulness of these techniques for barley cultivar discrimination was compared to a standard method, SDS-PAGE of hordeins; additionally, we investigated the reliability of glycoprotein 0173-083S/Y 1/0505-0323 $3.50+.25/0

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blotting for barley cultivar discrimination by examining to what extent the glycoprotein band patterns obtained are independent of environmental influences.

2 Materials and methods 2.1 Apparatus and chemicals All apparatus for horizontal electrophoresis (Multiphor 11, Miicrodrive I) and blotting (Novablot) were from Pharmacia-LKB (Freiburg, Germany). GelBond PAC film was from FMC (Rockland, ME, USA). Polyvinylidene difluoride (PVDF) membranes (Immobilon P) were from Millipore (Eschborn, Germany). Acrylamide (2 X crystallized) N,N-methylenebisacrylamide, SDS, gelatine (liquid), HRP (400 U/mg), Con A, and 3,3' diaminobenzidine (DAB) were from Serva (Heidelberg, Germany). Dansylhydrazine, polyvinylpyrrolidone (PVP, M, 40000), 4-chloro1-naphthol and a-D-methylmannoside were from Sigma (Deisenhofen, Germany). Periodic acid, bovine serum albumin (BSA, fraction V), and all other chemicals (analytical grade) for buffers and for electrophoresis were from Merck (Darmstadt, Germany). 2.2 Sample preparation 2.2.1 Seed material Samples of 20 European barley (Hordeum vulgare L.) cultivars were generously supplied by M. Baumer (Weihenstephan, Germany). Barley cultivars were predominantly from the 1988 harvest and grown in southern Bavaria near Weihenstephan. Additionally, samples of the cultivar ,,Franks" (harvested in 1986) were obtained from breeders from northern Bavaria. 2.2.2 Extraction of barley seed proteins Individual grains, or groups of five grains of each variety, were crushed and pulverized with a hammer until the powder passed a 1 mm sieve. (i) For extraction of albumins and globulins, the powder was mixed with five parts w/v of TrisHCl buffer (50 mM, pH 7.5) and extracted for 1 h at 4 "C with occasional vortexing. Then the suspension was centrifuged (20000 g , 20 min, 4°C) and the supernatant was mixed with an equal volume of SDS sample buffer (50 mM Tris-HC1,

pH 6.8,2% w/v SDS, 1O/o w/v dithiothreitol, 10% w/v glycerol and a trace of Bromophenol Blue dye), heated in a boiling water bath for 5 min, cooled to room temperature and, after adding 0.5 O/o w/v dithiothreitol, centrifuged (20000 g, 5 min, 20 "C).The supernatant was either used immediately for SDS-PAGE or stored at -20" until use. (ii) For the extraction of hordeins, the flour was mixed with four parts w/v of 55 O/o v/v aqueous 2-propanol and extracted for 2 h at 20 "C. Subsequently, the suspension was centrifuged (20000 g, 20 min, 20°C). The supernatant was then mixed with five volumes of SDS sample buffer and treated further as above. 2.3 SDS-PAGE All SDS-PAGE separations were performed on horizontal systems using thin gels cast on GelBond PAG film according to Gorg et al. [17].All experiments were performed in duplicate. Discontinuous [18] SDS gels (250 X 125 X 0.5 mm3) containing a linear acrylamide gradient (12-15 O/o T, 4 % C, 0.1% w/v SDS and 375 mM Tris-HCl, pH 8.8) and a stacking gel (4%T,4% C,O.lO/o w/vSDS, 125 mMTris-HC1, pH 6.8) were cast as described previously [19]. The electrode bufferwas25 mMTris, 192mMglycine,O.l %w/vSDS. Samples were applied into precast application slots (7 X 2 X 0.25 mm3).Proteins were stacked at 200 V/30 mA and resolved at 500 V/30 mA (total time approximately 3 h). When silver staining was performed 2 pL of each sample were applied, and 4 pL were applied when the separated proteins were blotted onto an immobilizing membrane. When the proteins were silver stained, the gels were fixed in methanol/acetic acid/water (4/1/5) for 1 h and stained with silver nitrate according to Blum etal. [20]; otherwise, the separated proteins were electroblotted onto a PVDF membrane immediately after electrophoresis without a fixation step. In the latter case, the gel was cast on the hydrophobic side of the GelBond PAG film (contrary to the usual procedure) to facilitate the removal of the gel from the plastic backing. Alternatively, a gel remover (Pharmacia-LKB, Freiburg, Germany) was used. 2.4 Electroblotting The electrophoretically separated proteins were electroblotted onto an immobilizing PVDF membrane using the semidry blotting technique of Kyhse-Andersen [21].Transfer was performed at 0.8 mA/cm* for 1 h at room tempera-

Table 1. Detection of glycoproteins after electrophoresis In the electrophoresis gel

Formation of aldehydes by periodate oxidation of vicinal hydroxyls and reaction of these aldehydes with

Specific detection of carbohydrate moieties using labeled lectins

-Schiff's reagent [4] -dansylhydrazine [S]

-radioactive label [6] -fluorescence label 171 -enzyme label [8,9]

After blotting onto an immobilizing membrane

Formation of aldehydes by periodate oxidation of vicinal hydroxyls and reaction of these aldehydes with

Specific detection of carbohydrate moieties with the help of lectins directly, using

indirectly, using

-enzyme hydrazides [l I] dansylhydrazine

-radioactive label 1121 -enzyme label [13] -fluorescence label [ 131

-antibodies against lectins 1131 -0ligomeric lectins +enzymes [14, 151 -avidin/biotin 1161

A

~:/t.cf,.o/JfIo,.=,Ssrs

1991, /2, 323-330

ture. N o cooling device was necessary because of the low Joule heat produced. After the electrophoretic transfer was terminated, the PVDF membrane was washed 4 X (10 min each) with PBS (50 mM phosphate buffer, pH 7.4,200 m M NaC1) to remove residual SDS that might interfere with glycoprotein staining. Then the membrane was air-dried and stored (sometimes several weeks) between two sheets of filter paper at 4°C in a sealed plastic bag until use. To check whether the electrophoretic transfer was performed correctly, a piece of membrane (2 cm wide) was cut off at one edge and stained with the general protein stain India ink

WI. 2.5 Detection of glycoproteins 2.5.1 Detection of glycoproteins by the periodate/ dansylhydrazine method The dried PVDF membrane was wetted in methanol for a few seconds and washed 2 X (5 rnin each) with PBS. Then the membrane was soaked in periodic acid (10 mM) in 100 mM sodium acetate buffer, pH 4.5, and gently rocked for 30 min at room temperature. It was again washed twice (5 min each) with PBS and then incubated with dansylhydrazine (0.01 Yo w/vin2%v/vaceticacid /2Oo/0v/vmethanol) in the dark for 1 h at room temperature.Next, the membrane was again washed twice (5 mill each) in PBS and dried. Glycoproteins were detected as fluorescent bands when illuminated with a long-wave UV lamp. Control experiments to exclude self-fluorescence of ceriain proteins or unspecific binding of dansylhydrazine to proteins were performed by omitting the dansylhydrazine and the periodic acid step, respectively.

2.5.2 Detection of Con A - binding glycoproteins The dried PVDF membrane was wetted in methanol and washed twice in TBS (50 mM Tris-HC1, pH 7.4, 200 m M NaCl, 1 mM CaCl,, 1 mM MnCl,). To saturate nonoccupied binding sites on the membrane, it was then either incuhated in 1Oo/ w/v gelatine, 1% w/v PVP or 1O/o w/v BSA (glycoprotein-free) in Tris buffered saline (TBS) for 6 h at room temperature. Glycoprotein-free BSA was obtained by periodate treatment of BSA according to Glass et al. [13]. Subsequently, the membrane was washed twice (5 min each) with TBS, followed by incubation with Con A (50 pg/mL) in PVP (0.5 O/o w/v in TBS) for 2 h or overnight at room temperature with gentle shaking. Then the membrane was washed four times ( 5 rnin each) with TBS and incubated with HRP (50 pg/mL) in PVP (0.5 O/o w/v in TBS) for 2 h at room temperature. Afterwards, the PVDF membrane was again washed four times ( 5 rnin each) in TBS with gentle shaking. The PVDF-bound glycoprotein-Con A-HRP complex was detected by staining with O.0lo/o v/v H,O, and 0.06% 4-chloro-1-naphthol [14] or 0.010/0 v/v H202 and 0.03 O/o w/v 3,3' DAB, respectively, in phosphate buffer (50 mM,pH 7.4). Incubation with the HRP substrate solutions was carried out for 3-5 min until the glycoprotein bands were clearly visible. Then the reaction was stopped by washing the membrane with distilled water. Unspecific binding of Con A was checked by adequate control experiments in which an inhibitory sugar (100 mM 4a-~-methyl-mannoside) was added to the Con A incubation buffer). Endogenous lectin-like activity or peroxidase activity was checked

Barley cultivar discrimination by glycoprotein blotting

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by omitting the Con A and the HRP incubation steps, respectively.

3 Results and discussion 3.1 Choice of the appropriate protein fractions In preceding experiments we investigated which protein fractions of barley seeds contain a high degree of glycoproteins. For this, barley seed proteins were fractionated on the basis of their solubility properties; these fractions were then characterized with respect to their glycoprotein composition by SDS-PAGE and lectin blotting. Using a modified extraction procedure according to Osborne [23], we obtained three protein fractions (albumins/globulins, prolamins and glutelins, respectively) by successively extracting barley meal with Tris-HC1 buffer, aqueous 2-propanol, and SDS buffer, respectively. Subsequently, the proteins of each fraction were electrophoretically separated by SDSPAGE and electroblotted onto an immobilizing membrane. Glucose- or mannose-containing glycoproteins were then specifically stained by the Con A/HRP method. Our results showed that the prevailing number of glycoproteins is located in the albumin/globulin fraction (Gorg eral. in preparation). For this reason, the protein fraction that is extractable with Tris-HC1 buffer (referred to as the albumin/ globulin fraction or Tris-buffer soluble fraction) was used for further investigations.

3.2 Optimization of the Con A/HRP staining method

In order to stain glycoproteins blotted onto a PVDF membrane as specifically and sensitively as possible by the Con A/HRP method, the individual steps of the staining procedure were optimized first. 3.2.1 Optimization of the

step

The aim of the blocking (or quenching) step is to saturate unoccupied binding sites on the immobilizing membrane in order to minimize unspecific background staining. Of the blocking agents frequently used in immunoblotting (gelatine, B SA and ovalbumin) egg-ovalbumin was excluded without testing its properties because it is a glycoprotein itself. Liquid gelatine was also not suitable in our hands because of heavy background staining. Commercially available BSA worked better, but the best results were obtained with glycoprotein-free BSA. Additionally, we also tested PVP, which gave excellent results, comparable to glycoprotein-free BSA. Because of the relatively high cost of BSAand the necessity of removing glycoprotein impurities from it by periodate oxidation (which is a time-consuming procedure), we used PVP as blocking agent almost exclusively during our further investigations.

3.2.2 Comparison of different colour reagents In our hands 4-chloro-1-naphthol (producing purple-colored glycoprotein bands) and 3,3' diaminobenzidine (giving brown glycoprotein bands) were about equal in sensitivity. In spot tests we were able to detect approximately 1 ng of egg-white albumin spotted onto a PVDF membrane, and

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comparable sensitivity was also observed after SDS-PAGE and electroblotting. However, unspecific background staining was less intense when using chloronaphthol instead of DAB. Further, DAB is occasionally claimed to be carcinogenic, whereas chloronaphthol is not, so that we preferred chloronaphthol. In an early stage of our experiments we also tried the tetrazolium staining method of Taketa [24], which turned out to be about twice as sensitive as the DAB and cbloronaphthol staining techniques. However, because of the relatively high cost of some of the chemicals required for this staining technique, we restricted its application to the detection of glycoproteins on two-dimensinal blots where the demand for high sensitivity is crucial (Gorg etal. in preparation).

3.2.3 Optimization of the incubation- and washing steps In our experience, detection limits can be lowered by prolonged incubation time with Con A and HRP, which allows more molecules to bind specificallyto the immobilized glycoproteins. On the other hand, unspecific binding to the membrane increases as well, resulting in intensified background staining. Removing this background staining requires additional washing steps, which may also remove specifically bound Con A/HRP from the glycoproteins because of the weak binding forces interacting between them. Summing up, prolonged incubation times can lead to a loss in sensitivity if unacceptably high background staining demands extensive washing. In practice, compromises usually have to be made to obtain a reasonable signal-to-noise ratio in combination with high sensitivity. In our hands, two hours’ incubation time with Con A and HRP gave best results. When longer incubation times were applied, the enhancement in sensitivity was almost negligible, but background staining increased with time. Nevertheless, incubation overnight was sometimes employed for practical reasons. Other critical factors were the quantity and duration ofwashing steps. Because of the relatively weak binding forces between Con A and the glycoproteins, in comparison to antigen-antibody or avidin-biotin binding forces, prolonged washing steps (>30 min) and/or vigorous shaking had 10 be avoided under all circumstances in order not to wash off the Con A/HRP complex from the immobilizing membrane, which would result in a loss of sensitivity (d[25]). Three to four washing steps, 5 min each, with gentle shaking gave best results. Other factors are the pH and temperature of the incubation buffers. Con A binds best to carbohydrates at pH 5 [26]; on the other hand, as it forms a tetrameric structure (which allows the simultaneous binding of immobilized glycoproteins and HRP) exclusively at neutral or basic pH, pH 7.4 was chosen as a compromise [14].Attention also needs to be paid to keeping the temperature of the incubation buffers constant during incubation steps since the association and dissociation rate of the Con A/HRP complex is temperature-dependent [25]. Laboratory shakers usually produce heat which can warm up buffers in insufficiently isolated incubation trays. Best results are obtained when using a shaking water bath at constant temperature, but an isolated incubation tray may work as well. Summing up, attention has to be paid to the pH and temperature of incubation- and washing solutions, as well as to the duration and number of

individual washing steps, in order to obtain reproducible results. All experiments described here were performed at least in duplicate or in triplicate. Highly reproducible protein and blot patterns were obtained when the conditions described in Table 2 were strictly followed; this table summarizes the optimized procedure for glycoprotein staining of barley seed proteins blotted onto a PVDF membrane.

3.3 Cultivar discrimination 3.3.1 Cultivar discrimination by SDS-PAGE of albumins/ globulins and silver staining When the albumins/globulins separated by SDS-PAGE were stained with silver nitrate, a multiplicity of protein bands was observed in the entire molecular mass range between 10 kDa and 100 kDa. However, the protein patterns obtained were more or less identical between the individual cultivars; the only exception was cv. “Triton” (No. 6), which showed a slightly deviating pattern (marked by an arrow; Fig. 1). Reasons for this are probably that the albumins and globulins are often enzymes whose amino acid sequences are highly conserved during phylogenesis: mutations in the amino acid composition large enough to be detected by SDS-PAGE would normally be lethal because the enzymes do not function properly. On the whole, barley cultivar discrimination on the basis of silver-stained albumin/ globulin patterns after SDS-PAGE is not promising.

3.3.2 Barley cultivar discrimination by glycoprotein staining using the periodateldansylhydrazine method When the same protein fraction (albumin/globulin fraction) was specifically stained for carbohydrate moieties with the periodate/dansylhydrazine reagent, the protein patterns obtained differed radically from the protein patterns obtained by silver staining (Fig. 2). Three major glycoproteins in the molecular mass range of 12,17, and 67 kDa were striking, as well as a 30 kDa band, which only occurred in cv. “Triton” (No. 6). Additionally, a multiplicity of weak, rather diffuse glycoprotein bands was distributed over the entire molecular mass range from 10 to 100 kDa. These minor glycoproteins, however, were not suited for barley cultivar discrimination, no more than the 12 kDa and 67 kDa major glycoproteins, because they exhibited identical Table 2. Optimized procedure for glycoprotein staining of barley seed proteins blotted onto a PVDF membranea’ ~

Step

Reagent

Time

Wash Blocking Wash Con A incubation Wash HRP incubation Wash Staining

TB S 1VoPVP in TBS TB S 50 pg/mL Con A in TBS-PVP TB S 50 pg/mL peroxidase in TBS-PVP TB S 0.01% Hz02 + 0.06% 4-chloro-lnaphthol in phosphate buffer (50 mM, pH 7.4) distilled water

2x5 rnin

Stou

6h 2 x 5 min 2h 4 x 5 min 2h 4x5 min 3-5 min

5 min

a) Abbreviations: TBS, Tris buffered saline (50 mM Tris-HCI, pH 7.4, 200 mM NaCI, 1 mM CaCI2,l mM MnC12);PVP, polyvinylpyrrolidone; Con A, concanavalin A: TBS-PVP, 0.5% polyvinylpyrrolidone in Tris buffered saline; HRP, horseradish peroxidase

i;lerrrophorrsls 1991, I , 323-330

Barley cultivar discrimination by glycoprolein blotting

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F‘igurc 1 , Horizontal SDS-PAGE of barley seed proteins extracted with Tris-HC1 buffer. Silver staining according to Blum e t a / . [20]. Barley cultivars: 1) Robur, 2 ) Dura, 3 ) Gerbel, 4) Hasso, 5 ) Tapir, 6) Triton, 7) Ogra, 8 ) Mammut, 9) Vogelsanger Gold, 10) Banjo, 11) Birgit, 12) Brunhild, 13) Franka, 14) Igri, 15) Marilyn, 16) Sonja, 17) Viola, 18) Diana, 19) Soiiate, 20) Gloria.

Figure 2. Detection of barley glycoproteins (Tris-HCI soluble) by the periodate/dansylhydrazine method after SDS-PAGE and blotting onto a PVDF membrane. Barley cultivars as in Fig. 1.

Figure3. (A) Detection ofbarley glycoproteiiis (Tris-HClextracts) by the C o n A / H R P stainingmethod after SDS-PAGEandblottingonto an immobilizing PVDF membrane (color reagent: 3,3’ diaminobenzidine); barley cultivars as in Fig. 1. (B) Total protein pattern of cv. “Gloria”(No. 20); blot stained with India ink.

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patterns in all of the cultivars examined. The only glycoproteins which showed differences between the individual cultivars were the 17 kDa band and the 30 kDa band of cv.“Triton” (No. 6). By taking the absence or presence of these bands as the only criterion for differentiation, irrespective of their quantitative expression, the 20 cultivars examined could be arranged into three groups: group I (17 kDa band present),which contains 15 cultivars; group I1 (17 kDa band absent), which contains four cultivars; and group I11 (30 kDa band present), which contains only one cultivar. In order to exclude artifacts caused by unspecific binding of dansylhydrazine to glycoproteins (e.g., by hydrophobic interactions), we performed several control experiments in which the PVDF membrane was incubated with dansylhydrazine alone, without a preceding oxidation step. These experiments resulted in largely diminished binding of dansylhydrazine to the glycoproteins in comparison to those detected by the staining protocol described (results not shown). We assume that the weak binding observed is caused by free, terminal carbonyl groups that are able to react with dansylhydrazine.

Electrophorrsis 1991, 12, 323-330

3.3.3 Barley cultivar discrimination by glycoprotein staining with the Con A/HRP method When the PVDF membrane was stained for glycoproteins with the Con A/HRP reagent, three groups of major glycoproteins were detected (Fig. 3): a 17 kDa glycoprotein that was also stained by the periodate/dansylhydrazine reagent; two neighboring bands with M,’s in the range of 39-41 kDa; and a quadruplet in the molecular mass range from 55-67 kDa that showed differences in the intensities of band expression between the individual cultivars. Furthermore, five cultivars (Nos. 14,16,17,19, and 20) exhibited a 45 kDa band with intermediate intensity; cv. “Triton” (No. 6) showed an additional 30 kDa band. Finally, about a dozen minor bands with molecular masses between 10 and 100 kDa were found in all cultivars examined. When comparing the glycoprotein band patterns obtained with the periodate/dansyl hydrazine reagent and the Con A/HRP reagent, not only similarities but also differences between the individual glycoprotein patterns coud be detected: (i) The 17 kDa band patterns were identical in both cases. (ii) The 12 kDa band was only a minor glycoprotein when

figure 4. Effect of environmental conditions on the glycoprotein banding pattern. Single-seed analysis of cv. “Franka” (No. 13), grown in two different areas of Bavaria and harvested in 1986 (a) and 1988 (b). (A) Glycoprotein staining by the Con A/HRP method after SDS-PAGE and blotting onto a PVDF membrane (color reagent: 4-chloro-1-naphthol). (B) Total protein pattern (blot stained with India ink),

Figure 5. Horizontal SDS-PAGE of barley prolamins (hordeins) extracted with 55 Barley cultivars as in Fig. 1.

aqueous 2-propanol. Silver staining according to Blum era/. [20].

Llectrophoresis 1991,

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stained with Con A/HRP; however, it is a major glycoprotein when stained with periodate/dansylhydrazine. This glycoprotein obviously contains only few binding sites for Con A, suggesting a low glucose and mannose content. (iii) The 30 kDa glycoprotein band of cv. “Triton” (No. 6) was detected by both techniques with comparable sensitivity. (iv) The 39-41 kDa doublet and the 55-67 kDa quadruplet were well stained with Con AIHRP, but only weakly stained with periodate/dansylhydrazine (with one exception); this exception was the uppermost band of the 55-67 kDa quadruplet, which was also stained well with dansylhydrazine. These bands were probably recognized better by Con A/HRP because of the greater sensitivity of this highly specific, enzyme-enhanced technique in comparison to the more “general”,but less sensitive, dansylhydrazine method.

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only the B (30-45 kDa) and C hordeins (49-72 kDa) were utilized because only these exhibited protein polymorphism, while the A hordeins (< 25 kDa) showed identical protein patterns. The combined B and C hordein patterns allowed us to classify the 20 cultivars into nine groups. The largest group contains ten cultivars (Nos. 4,5,7, 10,14-17, 19, and 20), two groups contain two cultivars each (Nos. 1 and 3, as well as 8 and 13), and six groups contain only one cultivar (Nos. 2,6, 9, 11, 12, and 18). 3.3.5 Grouping of cultivars according to glycoprotein and hordein patterns

On the basis of glycoprotein staining with Con A/HRP it was possible to classify the 20 barley cultivars into six groups, the largest of which contains twelve cultivars, while When staining the PVDF membrane-bound glycoproteins the results of SDS-PAGE of hordeins enabled us to divide with the Con A/HRP reagent, six different banding patthe cultivars into nine groups consisting of 1-10 cultivars. terns were obtained - if one neglects differences in glycoThe combined use of glycoprotein and hordein patterns protein band intensities and takes into account only the abmade it possible to classify the cultivars into twelve groups, sence or presence of certain glycoprotein bands (the 17,30, the largest of which contains only four cultivars, which is a and 45 kDa bands) for reasons to be discussed later. Three considerable increase in resolution. Group I contains four barley cultivars (Nos. 2, 6, and 15) could be distin~iii~hed cultivars (Nos. 4,5,7, and lo), Group I1 contains three cultiunequivocally, whereas the others could be classified into vars (Nos. 16, 19, and 20), and groups 111-V two cultivars three groups containing twelve (Nos. 1,3-5,7-13, and 18), each (Nos. 1 and 2,8 and 13,14 and 16, respectively); seven three (Nos. 16,19, and 20) and two cultivars (Nos. 14 and cultivars (Nos. 2,6, 9, 11, 12, 15, and 18) can be identified 17). uniquely. Single grain extraction was used for purity control of a breeding line, whereas flour samples were used to establish protein patterns typical of each variety in order to exclude anomalies that might have been caused by not absolutely homogeneous seed material.To find out whether the glycoprotein band patterns are influenced by environmental factors, we performed multiple single-seed analyses. For this purpose we used seeds ofcv.“Franka”(No. 13) that was harvested in different areas of Bavaria in 1986 and 1988.Ourresults showed that the band patterns were uniform (with one exception), but differed in their band intensities (Fig. 4). For this reason we do not recommend the use of quantitative differences in the glycoprotein band patterns, but solely of qualitative differences (i. e., absence or presence of certain glycoprotein bands), for barley cultivar discrimination.The exception mentioned above may have been the result of seed material that was not absolutely homogeneous. In control experiments in which the inhibitory sugar a-D-methylmannoside was added to the Con A incubation buffer, in order to investigate whether any unspecific binding reactions had taken place, the binding of Con A to the membrane-bound glycoproteins was inhibited completely; control experiments to detect possible lectin-like activity of endogeneous peroxidase activity also showed negative results,indicating that the detection of glycoproteins with Con A/HRP is a highly specific reaction (results not shown). 3.3.4 Barley cultivar discrimination by SDS-PAGE of hordeins and silver staining In order to decide whether the glycoprotein staining technique is useful for barley cultivar discrimination, we compared its discriminating power to that of a standard method, SDS-PAGE of hordeins (Fig. 5). For cultivar discrimination

4 Concluding remarks Glycoprotein blotting is not an alternative, but rather a valuable supplement, to SDS-PAGE of hordeins for barley cultivar discrimination. A combined use of both techniques allows much better differentiation than either method taken alone. Staining of the blotted glycoproteins with Con A/HRP is superior to staining with periodate/dansylhydrazine due to its greater sensitivity and specificity.The latter, however, is cheaper and easier to perform.The sensitivity of the Con A/HRP method is comparable to the staining of proteins on blots with India ink. Possible future developments in this field may be double (or multiple) staining techniques for glycoproteins, where lectins with different carbohydrate specificities could be applied simultaneously on the same blot in combination with enzymes (and substrates) that produce various colored glycoprotein band patterns. Whenever this technique can not be used (either because some lectins are glycoproteins themselves, leading to cross reactions, or because of different pH or ion requirements of the different enzymes), the procedure can be modified such that the lectins (e.g. Con A) are released from the membrane by an inhibitory sugar (e.g. a-D-methyl-mannoside) after staining. The membrane is then incubated with a lectin of different sugar specificity and stained a second time. In this case it would again be possible to obtain different glycoprotein band patterns on a single blot. Thus, by extending the spectrum of lectins, cultivar discrimination would be further improved. The authors wish to thank Dr. M. Baumerand Mr. R . Grauf of the Bayerische Landesanstalt f u r Bodenkunde und Pflanzenbau, Freising- Weihenstephan, for generously providing seed samples and for valuable discussions. November 23, 1990

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W. Weiss, W. Postel and A . Gorg

Elrcrrophorrsis 1991, 12. 3311-337

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Walter Weiss Wilhelm Postel Angelika Gorg Lehrstuhl fiir Allgemeine Lebensmitteltechnologie, Technische Universitat Miinchen, Freising-Weihenstephan

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Barley cultivar discrimination: 11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-1 1 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitolsoluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.

1 Introduction Barley (Hordeum vulgare L.) is the fourth most extensively grown cereal on the globe, and in some countries of the cool, temperate zone its production exceeds even that of wheat [l].Barley is generally grown in the form of tworowed or six-rowed cultivars. In Germany, two-rowed

Correspondence: Priv. Doz. Dr. A. Gorg, Institut fur Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen-Weihenstephan, DW-8050 Freisiog-Weihenstephan, Germany Abbreviations: Bis, N,N-methylenebisacrylamide; CV., cultivar; DTT, dithiothreitol; IEF,isoelectric focusing; IPG,immobilized p H gradient; M,, relative molecular mass; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TEMED, N,N,N,N-tetramethylethylenedianiine; Tris, Tris(hydroxymethy1)aminomethane

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

spring barley cultivars are used for malting and brewing, whereas two- and six-rowed winter barleys are mainly grown for animal feeding. Because of the different processing properties of the cultivars, their correct identification is important to the malting and brewing industries. Although barley cultivars are often identified by grain morphological characteristics (e. g. rachilla hair length, aleurone color), many cultivars cannot be distinguished by this means. For this reason, supplementary techniques for identification are needed. One of the most powerful techniques is electrophoresis of the aqueous alcohol-soluble endosperrn proteins, the prolamins (known as hordeins), and, to a lesser extent, electrophoresis of albumins and globulins. Identification of cultivars by protein electrophoresis is possible because the electrophoretic patterns of storage proteins are cultivar-specific and independent of environmental conditions [l, 21. Usually, electrophoresis of hordeins is per-