Electrophoresis 1990, 11, 123-128
Fixing and staining of gliadins
181 Sandeen, G., Wood, W. I. and Felsenfeld. G.. Nucleic Acids Res. 1980,8,3757-3778. 191 Bassuk, J. A. and Mayfield, J . E., Biochemistr?, 1982.21.1024- 1027. [ l o ] Levy-Wilson, B., Denker, M. S. and Ito, E.. Biochemislry 1983.22, 171 5-1 721. I 1 1 I Katula, K. S.. Developmental Biol. 1983. 98. 15-27. [I21 Johns, E. W., The HMG Chromosomal Proteins. Academic Press, London 1982, pp. 1-251. 1131 Einck,L.andBustin,M.,Exp. CellRes. 1985,156,295-310. [ 141 Blobel, G. and Potter, V. R., Science 1966 154, 1662-1665. [ 151 Bradford, M. M., Anal. Biochem. 1976, 72,248-254. 1161 Samal, B. B., Anal. Biochem. 1987,163,42-44. 1171 Neuhoff, V., Stamm, R. and Eibl, H., Electrophoresis 1985. 6 , 427-448. 1 181 Mayes, E. L. V. and Johns, E. W., in: Johns, E. W. (Ed.). The HMG Chromosomal Proteins, Academic Press, London 1982. pp. 223-247.
Robert L. Clements USDA-ARS Soft Wheat Quality Laboratory, Wooster, OH
123
1191 Landsman, D. and Bustin, M., J. B i d . Chem. 1986. 261,
16087- I609 1. 1201 Srikantha, T., Landsman, D. and Bustin, M., J . B i d . Chern. 1988. 263. 13200-13503. [21 I Merril. C. R., Goldman, D. and Van Keuren, M. L., Electrophoresis 1982,3,17-23. I22 I Mayes, E. L. V., in: Johns, E. W. (Ed.), The HMG Chromosomd Pro teins, Academic Press, London 1982, pp. 9-40. 1231 Seyedin, S. M. and Kistler, W. S., J . Biol. Chem. 1979. 254. 11264-1 1271. 1241 Gordon. J . S., Rosenfeld, B. I., Kaufman, R. and Williams. D. L., Biochemistry 1980. 19.4395-4402. [251 Shastri, K., Isackson, P. J., Fishback, J . L., Land, M. D. and Reeck, G. R., Nucleic Acids Res. 1982,10, 5059-5072. [261 Chiva, M. and Mezquita. C., FEBS Lett. 1983, 162. 324-328. 1271 Rabbani, A,, Goodwin, G. H. and Johns, E. W.. Biochem. J. 1978, 173,497-505.
Alternative methods for fixing and staining gliadins in polyacrylamide gels Staining efficiencies ofCoomassieBrilliant Blue G-250(CBB G-250) and Coomassie Brilliant Blue R-250 (CBB R-250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R-250 in 12 % TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low- and highmobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G-250 and R-250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G-250 in 2 % TCA, in 2 % T C A containing 5 % sodium sulfate, and in 2 % and 5 % phosphoric acid containing 5 % sodium chloride or 5 % sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R-250 in 12 % TCA.
1 Introduction Polyacrylamide gel electrophoresis (PAGE) of gliadins (“gliadin fingerprinting”) has become an invaluable technique for identifying wheat genotypes and for establishing homogeneity. Although extraction procedures and electrophoretic conditions vary considerably among laboratories, staining procedures employing Coomassie Brilliant Blue R-250 (CBB R-250) are used almost universally, and use of CBB R-250 in 12 % trichloroacetic acid (TCA) has become virtually standard [ 1-41. Some procedures also call for prefixation and/or washing in 12 % T C A [4]. Employment of CBB R-250 with T C A as fixative and dye carrier for gliadin P A G E is a natural consequence of successful application of the procedure to Correspondence: Dr. Robert L. Clements, USDA-ARS Soft Wheat Quality Laboratory, Wooster, OH 44691, USA Abbreviations: CBB, Coomassie Brilliant Blue; PAGE, polyacrylamide gel electrophoresis; TCA, trichloroacetic acid Mention of firm name or product does not constitute endorsement by the U S . Department of Agriculture. 0 VCH Veriagsgeseilschaft mbH, D-6940 Weinheim, 1990
other proteins. This procedure takes advantage of the effectiveness of T C A as a universal protein precipitant. The introduction of high concentrations of T C A (12.5 %)was a result of efforts to optimize fixation and staining with minimum background [ 5 , 61. Under these conditions the dye is in a colloidal state, and although capable of binding to protein, it is essentially insoluble in the medium; background therefore is negligible. The use of Coomassie dyes for staining proteins in gels in T C A and other media has been summarized in several reports [7-91. CBB R-250/12 % T C A also provides good results as a gliadin fixative and stain. However, the gliadins, as prolamines, represent a class of proteins that exhibit unique solubility properties. They are essentially insoluble in aqueous buffers and salt solutions that disperse albumins and globulins. On the other hand, the gliadins are soluble in alcohols and weak acids, agents that precipitate and/or denature many proteins. The CBB R-250112 % T C A procedure has been used extensively in this laboratory for routine fixation and staining of gliadins separated by P A G E [ 10-121. However, with continued use of the technique, several drawbacks have become 01 73-0835/90/0202-0123%2.50/0
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evident: TCA is relatively expensive:, and as used in this laboratory (1 2 % T C A for fix/stain only, and not for prefixation or washing) has accounted for about 75 % of the cost of reagents used for PAGE. Also, T C A is extremely corrosive and caustic, and the large quantities required for routine use in 12 % solutions pose a risk to personnel and equipment. Because aqueous concentrated T C A decomposes on standing, 12 % solutions must be prepared just before use. Moreover, decomposition products of TCA include chloroform, creating a potential disposal problem.
However, proteins employed in the study were albumins and globulins. Also, their recommended procedures all included prefixation in 12 % TCA. Several additives (e. g., salts) were included in evaluations of various media. The authors concluded that best staining occurs when conditions are adjusted so that the dye is in a colloidal form which does not precipitate. The unique behavior of the gliadins suggests that nonconventional approaches might be employed for fixation. The studies by Neuhoff et al. (91 show that the Coomassie dyes can function in a wide range of media if conditions result in the desired colloidal state. The objectives of the following study were to investigate alternative media for fixation and staining of gliadins in polyacrylamide gels, and to determine suitability of the media as carriers for CBB G-250 and CBB G-250dyes. A specific objective was to reduce or eliminate requirements for T C A in the media.
Recently, Neuhoff et al. [91 reported results of a systematic and detailed study of protein staining in polyacrylamide gels with R-250 and G-250 dyes. The authors included several media in their study, and concluded best results were obtained with CBB G-250 in TCA, perchloric acid or phosphoric acid.
Table 1.Effects ofstaininggliadinsinpolyacrylamidegel using CBB G-250 and R-250in variousmedia ~
Medium Acidic 5 % Acetic acid 5 % Acetic acid 5 % Acetic acid 10 % Acetic acid 1.2 % Picric acid (satd.) 1.2 % Picric acid (satd.) 1.2 YOPicric acid (satd.) 1.2 % Picric acid (satd.) 2 % Phosphoric acid 2 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 2 % TCA 2 % TCA 2 % TCA 2 % TCA 2 %TCA 2 %TCA 5 % TCA 12 % TCA Basic 5 % Sodium bicarbonate 5 % Sodium bicarbonate 5 Yo Sodium bicarbonate 10 % Sodium bicarbonate 10 % Sodium carbonate 10 % Sodium carbonate 10 % Sodium carbonate Neutral Water, deionized Water, deionized Water, tap Water, tap 5 % Sodium chloride 5 % Sodium sulfate 0.1 M Sodium picrate 0.1 M Sodium picrate
Salt
5 % NaCl 5 % Na,SO, 10 % NaCl 5 % NaCl 10 % NaCl 5 % NaCl 5 % NaCl
5 % NaCl 5 % NaCl 5 % Na,SO,
5 % NaCl 10 % NaCl 10 % NaCl 5 % Na,SO, 5 % NaCl
10 YONaCl
5 YONaCl 10 % NaCl
~
~~~~
a) Dye
b) Intensity
C) Background
d) Slow bands
e) Fast bands
G G G G G R G G G R G R G R G G R G G R G G R
3 2 2 2 2 2 3 2 3
2 3 2 3 2 2 2 2 3 3 3
2 3 3 3 2 2 3 3 3
2 1 2 2 3 3 3 3 3 3 2 2 3 3 3 3 3 2
1
3 3 3 1
3 3 2
3 3 1
3 3 3
G R G G G G G
2
G R G R G G G R
2 2 3 3 2 2
1
2 2 3 1 1
1 1
2 3 3 3 3 3 3 3 3 3 3 3
1
2 2 3 2 3 3 3 3 3 2 3 3 3
2 2 2 2 2 3 3
2 2 3 3 3
2
1 2 3 2 3 3 2 2
3 3 3 3
2 2 1
1 1
a) G, CBB G-250; R, CBB R-250 b) Overall intensity of stain on scale of 1 (very weak) to 3 (intense) c) Background on scale of 1 (heavy) to 3 (negligible) d) Fixation and intensity of slow bands on scale of 1 (very weak) to 3 (all fixed and fully stained) e) Fixation of fast bands on scale of 1 (very diffuse) to 3 (sharp)
1
2 3 2 3 1
1 1
2 2 1 1 2 2 2 2 2 2 2 1
Electrophoresis 1 9 9 0 , I I , 123-128
2 Materials and methods 2.1 Chemicals Acrylamide was “suitable for electrophoresis” grade from Sigma (St. Louis, MO). Dyes were CBB G-250 from J. T. Baker Chemical Co. and CBB R-250 (Serva Blue R) from Serva Fine Biochemicals (Westbury, NY). Other reagents were reagent grade chemicals from various sources. None of the reagents were further purified. Unless otherwise noted, water was double-deionized with a specific conductivity ofless than 0.3 mS/cm. T a p water, where specified, had a specific conductivity of about 700 mS/cm.
2.2 Electrophoresis The electrophoresis procedure, employing 16 x 18 cm x 1.5 mm gels containing 12 % total acrylamide with 3 % crosslinkage (12 % T, 3 % C ) and buffered with acetic acid, was described previously L 121. Wheat meals were extracted with ethylene glycol(2 mLper g meal) [ 101andextracts were stored at -20 OC. The same set of extracts was applied repeatedly to gels to provide replicate gels for staining studies. Loads were 8 pL per well on 10-well gels.
2.3 Staining and washing
Fixing and staining of gliadins
125
staining may have contributed to the loss of sharpness and resolution. Because of the effects in these two regions, media were evaluated for “slow band” and “fast band” staining, in addition to overall stain intensity and background. These four attributes were judged on a scale of 1 (poor) to 3 (excellent), with CBB R-250 in 12 % T C A serving as a standard(Tab1e 1). A dye-medium system with a combined score of 12 was thus judged to be as good as the standard. Although subjective, a score provides an indication of the potential usefulness of a particular system. However, the scores do not take into account such attributes as time required for staining or destaining, or stability of the stain. As noted earlier, staining efficiencies of CBB G-250 and CBB R-250 are a function of the colloidal state of the dye [7-91. Generally, acidic systems are employed for protein fixation and staining, and in such systems colloidal state is determined by strength and concentration of the acid and by the nature and concentration of any added electrolytes [9]. Optimum conditions are not the same for CBB G-250 and CBB R-250, however. Also, conditions that are optimum for staining may not provide fixation of all proteins. Experiments with simple acid systems showed that CBB R-250 in 5 %phosphoric acid resulted in excellent overall stain and low background, but gave weak slow bands and diffuse fast bands (Fig. lc). Saturated picric acid gave similar results, but with higher background (Fig. Id). Staining with CBB G-250 or CBB R250 in 5 % or 10 % acetic acid (not shown) resulted in poor fix-
Upon completion of electrophoresis, gels were transferred immediately to the fixative (400 mL). Dye (3 mL 0.5 % in 50 % aqueous ethanol) was then added dropwise with agitation. Additional dye was added in 18-20 h if concentration appeared to be limiting. Gels were agitated periodically during staining and were stained for different periods, depending upon the rate of staining. When stain intensity appeared to have attained a stable level (2-4 days), staining solution was decanted and the gel was washed. In most instances, avery short rinse (< 1min) with aqueous 50 % methanol110 % acetic acid followed by a second short rinse with aqueous 7 % acetic acid15 % methanol sufficed. Gels stained with CBB R-250 (and a few gels stained with CBB G-250) required slightly more washing, but no gels were destained other than to remove residual medium and precipitated dye. Gels were photographed immediately after washing unless otherwise noted. (The two outer lanes on each gel were deleted from the photographs). Results were evaluated by visual comparison of stained gels with gels stained with CBB R-250 in 12 % TCA.
3 Results and discussion 3.1 Comparison of different staining conditions Comparisons of gels stained under the various conditions showed qualitative differences, as well as differences in overall stain intensity and background, i. e., all bands were not affected in the same way. The low-mobility gliadins or ogliadins [ 131, in particular, were differentially fixed and/or stained. Some bands in this region were extremely weak or lacking in many gels in which the more mobile gliadins appeared to be fully fixed and stained. Also, those gliadins of moderate to high mobilities (including the a- and P-gliadins) showed differential effects, evident as diffused bands. Although appearing to be caused by incomplete fixation, poor
Figure 1 . Gliadins stained in 12 Yo polyacrylamide gels with Coomassie Brilliant Blue R-250 in (a) 12 % TCA, (b) 2 o/o TCA (c) 5 % phosphoric acid, and (d) saturated picric acid. Cultivars (left to right, all gels): Logan, Caldwell, Cardinal, Compton, Hart, Pike, Titan, Tyler.
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Electrophoresis 1990, 11, 123-128
Figure 2. Gliadins stained in 12 % polyacrylamide gels with Coomassie Brilliant Blue G-250 in acidic media: Effects of adding sodium chloride and sodium sulfate (vertical axis) to 5 % acetic acid, 5 % phosphoric acid and 2 Yo TCA (horizontal axis). Cultivars (left t o right, all gels): Logan, Caldwell, Cardinal, Compton, Hart, Pike, Titan, Tyler.
ation and staining. On the other hand, CBB R-250 in 2 % T C A (Fig. lb) gave results comparable to those obtained with CBB G-250 in 12 % TCA (Fig. 1a), with no apparent deficiencies in fixation.
but resulted in some diffusion of the fast bands, particularly in 2 % TCA. Incorporation of 5 % sodium sulfate in the three acid systems provided good fixation and staining of all bands. Similar results were obtained with saturated picric acid (not shown).
3.2 Addition of electrolytes
Experiments with neutral and basic salt solutions indicated gliadins are fixed in such media (as evidenced by turbid bands), but the conditions may not promote staining. CBB G-250 provided good staining under alkaline conditions (5 %sodium bicarbonate and 5 % sodium carbonate in Fig. 3a and b). However, CBB G-250 in 5 % sodium chloride or 5 % sodium sulfate produced very weak stains (Fig. 3c and d). Stephano etal. [14] have suggested sodium picrate (pH 7.0) as a medium for staining polypeptides in gels. However, neither CBB G-250 nor CBB R-250 provided satisfactory staining of the gliandins in this medium (Table 1).
Addition of electrolytes to acidic systems not only influences the colloidal state of the dye, but also affects fixation of gliadins. Fig. 2 shows effects of adding 5 ?41 sodiumchlorideor 5 % sodium sulfate to 5 % acetic acid, 5 % phosphoric acid and 2 % TCA, with CBB G-250 as the dye. Although the slow bands appeared to be fully fixed and stained in 2 % T C A without added salt, many of these bands were weakly stained in gels in 5 % acetic acid or 5 % phosphoric acid. Addition of 5 % sodium chloride greatly improved staining ofthese bands,
Electrophoresis 1990,11, 123-128
Figure 3. Gliadins stained in 12 Yo polyacrylamide gels with CBB G-250 in basic and neutral salt solutions: (a) 10 % sodium bicarbonate. (b) 10 % sodium carbonate, (c) 5 YOsodium chloride, and (d) 5 % sodium sulfate. Cultivars (left to right, allgels): Logan, Caldwell, Cardinal. Compton. Hart, Pike, Titan, Tyler.
3.3 Staining in water The results suggested the gliadins can be fixed and stained under a wide range of conditions, including relatively mild conditions. Therefore, experiments were performed using water alone as amedium. Goodresults were obtained with both CBB G-250 and CBB R-250 in tap water (Fig. 4), but qualitative differences were noted in patterns from the two dyes. In gels stained with CBB G-250, the slow bands were fixed and stained, but the fast bands were moderately diffused; in gels stained with CBB R-250, the slow bands were only partially stained but the fast bands were relatively sharp and intensely stained. In deionized water, CBB G-250 gave moderately stained patterns in which the slow bands were almost imperceptible. CBB R-250 produced relatively strong patterns, but as in gels stained with tap water, several slow bands were lacking. The behavior of the dyes in deionized water suggests the dyes alone may be capable of fixing (i. e., precipitating) some gliadins. Of the four water systems, only CBB G-250 in tap water resulted in staining of the slow-moving doublet noted in all eight cultivars.
3.4 Other staining systems Several systems in addition to those summarized in Table 1 were studied. Those described were included to illustrate
Fixing and staining of gliadins
127
Figure 4. Gliadins stained in 12 % polyacrylamide gels with CBB G-250 (left) and CBB R-250 (right) in tap water (top) and deionized water (bottom). Cultivars (left to right, all gels): Logan, Caldwell, Cardinal. Compton, Hart. Pike, Titan, Tyler.
systems that appear to be potentially useful, as well as to show quantitative and qualitative effects of various media on fixation and staining. Among media studied were ammonium sulfate solutions and acidic systems containing ammonium sulfate, including the 2 % phosphoric acid/6 % ammonium sulfate system of Neuhoff et al. 191. Results from these media were similar to those obtained with sodium chloride as salt, i. e., gels exhibited some degree of diffusion of the fast bands. The results suggest many other systems could provide acceptable fixation and staining of the gliadins. Requirements for fixation appear to be minimal, permitting adjustment of conditions to provide optimum staining. Several systems were judged to be comparable to the standard CBB R-250 in 12 % TCA. Among these were CBB G-250 in 2 % TCA, 2 % TCA containing 5 % sodium sulfate, and 2 % and 5 % phosphoric acid containing 5 % sodium chloride or 5 %sodium sulfate. In addition to reducing or eliminating TCA, these systems offer other advantages. CBB G-250, in these media, provides a highly transparent solution that permits easy observation during staining. Background is negligible, and no destaining is required (other than rinsing to remove precipitated dye). Stability of stain, as such, was not studied, but patterns appeared to be as stable as those obtained with CBB R-250 in 12 % TCA. The current method of choice in this laboratory is CBB G-250 in 2 % TCA/5 %sodium sulfate. Although the sodium sulfate is not essential, the salt appears to promote sharper bands and cleaner, stronger gels.
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Electrophoresis 1990,11, 128-133
The system has been applied routinely to more than 100 gels with excellent results. Gels may be allowed to remain in the staining solution for extended periods without deterioration of patterns. Stained gels wrapped in polyethylene film and stored for several months at 4 "C retained strong patterns. Storage for shorter periods in 5 % sodium sulfate at room temperature has also given good results.
4 Concluding remarks Staining gliadins with CBB R-250 in 12 c% TCA has become universal, primarily because the method provides good results. However, under some circumstances, it may be desirable or necessary to reduce or eliminate requirements for TCA. It appears that requirements for gliadin fixation are minimal, and that concentrated TCA is necessary only to provide suitable conditions for the staining process. Substitution of CBB G-250 for CBB R-250 permits staining in media other than 12 % TCA, and can provide results comparable to those obtained with CBB R-250 in 12 % TCA. Evaluation of results, together with comparisons of cost, convenience and other attributes may show that an alternate procedure offers specific advantages without sacrificing staining el'ficiency.
5 References [ 1I Autran, J. C., Bushuk, W., Wrigley, C. W. andzillman. R. R.. Cereal
Foods World 1979,24,471-472. 121 Cooke, R. J., Electrophoresis 1984,5,59-72. 131 Lookhart, G. L., Jones, B. L., Hall, S. B. and Finney. K. F.. Cereal Chem. 1982,59, 178-181. 141 Wrigley, C. W., Autran, J. C . and Bushuk, W., Adv. Cereal Sci. Technol. 1982,5,211-259. IS1 Chrambach, A., Reisfeld, R. A., Wyckhoff, M. and Zaccari.J..Anal. Biochem. 1967,20,150-154. 161 Rodbach, D. and Chrambach, A.,Anal.Biochem. 197 1.40.95-1 34. [71 Wilson, C. M., Methods Enzymol. 1983,91, 263-278. 181 Reisner, A. H., Methods Enzymol. 1984,104,439-441. [91 Neuhoff, V., Stamm, R. and Eibl, H., Electrophoresis 1985, 6. 427-448. [ 101 Clements, R. L., Cereal Chem. 1987,64,442-448. [ 11 I Clements, R. L., Cereal Chem. 1988,65, 150-152. I121 Clements, R. L., Electrophoresis 1988,9,90-93 1131 Kasarda, D. D., Bernardin, J. E. and Nimmo, C. C..Adv. Cereal. Sci. Technol. 1976,1, 158-236. 1141 Stephano, J. L., Gould, M. and Rojas-Galicia, L., Anal. Biochem. 1986,I52,308-313.
Received July 28, 1989
Patrick Masson' David M. Arciero2 Alan B. Hooper2 Claude Balny3 'Centre de Recherches du Service de Santedes Armees, Unite de Biochimie, La Tronche *Departmentof Genetics and Cell Biology, University of Minnesota, Saint Paul, MN 31nstitut National de la Sand et de la Recherche MBdicale, Unit6 128, B.P. 505 1, Montpellier
Electrophoresis at elevated hydrostatic pressure of the imultiheme hydroxylamine oxidoreductase The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 2 5 "C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.
1 Introduction Hydroxylamine oxidoreductase (HAO; EC 1.7.3.4) is a multiheme protein from the aerobic nitrifying bacterium Nitrosomonas [ 11 which catalyzes the oxidation of hydroxylamine to nitrite. H A 0 is a multimer of an a-subunit (- 63 kDa) containing at least 6 c-hemes and one active site which contains a
Correspondence: Dr. Patrick Masson, CRSSA, Unite de Biochimie, B.P. 87, F-38702 La Tronche Cedex, France Abbreviations: BSA, bovine serum albumin; HAO, hydroxylamine oxidoreductase; SDS, sodium dodecyl sulfate 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
heme-like structure, P-460 [2,3]. H A 0 can be isolated with a second 11 kDa c-cytochrome (referred to as a "P-subunit") which is not necessary for activity and is not present in current preparations of the pure enzyme 141.Thenumber of asubunits in the enzyme is not known. The molecular size, approximately 180-200 kDa [3, 51 based on hydrodynamic measurements, is uncertain since the shape of the protein is unknown. Treatment of H A 0 with sodium dodecyl sulfate (SDS) and then SDS/mercaptoethanol results in bands of progressively higher apparent molecular weight (125, 195 and 225 kDa) as seen on SDS-polyacrylamide gels [ 3 ] .The 63 kDa monomer has thus far been observable only after chemical removal of all c heme. This behavior in chemical denaturants has suggested polymerization and/or denaturation of the H A 0 monomer. 0 173-0835/90/0202-0128 $2.50/0
Fixing and staining of gliadins
181 Sandeen, G., Wood, W. I. and Felsenfeld. G.. Nucleic Acids Res. 1980,8,3757-3778. 191 Bassuk, J. A. and Mayfield, J . E., Biochemistr?, 1982.21.1024- 1027. [ l o ] Levy-Wilson, B., Denker, M. S. and Ito, E.. Biochemislry 1983.22, 171 5-1 721. I 1 1 I Katula, K. S.. Developmental Biol. 1983. 98. 15-27. [I21 Johns, E. W., The HMG Chromosomal Proteins. Academic Press, London 1982, pp. 1-251. 1131 Einck,L.andBustin,M.,Exp. CellRes. 1985,156,295-310. [ 141 Blobel, G. and Potter, V. R., Science 1966 154, 1662-1665. [ 151 Bradford, M. M., Anal. Biochem. 1976, 72,248-254. 1161 Samal, B. B., Anal. Biochem. 1987,163,42-44. 1171 Neuhoff, V., Stamm, R. and Eibl, H., Electrophoresis 1985. 6 , 427-448. 1 181 Mayes, E. L. V. and Johns, E. W., in: Johns, E. W. (Ed.). The HMG Chromosomal Proteins, Academic Press, London 1982. pp. 223-247.
Robert L. Clements USDA-ARS Soft Wheat Quality Laboratory, Wooster, OH
123
1191 Landsman, D. and Bustin, M., J. B i d . Chem. 1986. 261,
16087- I609 1. 1201 Srikantha, T., Landsman, D. and Bustin, M., J . B i d . Chern. 1988. 263. 13200-13503. [21 I Merril. C. R., Goldman, D. and Van Keuren, M. L., Electrophoresis 1982,3,17-23. I22 I Mayes, E. L. V., in: Johns, E. W. (Ed.), The HMG Chromosomd Pro teins, Academic Press, London 1982, pp. 9-40. 1231 Seyedin, S. M. and Kistler, W. S., J . Biol. Chem. 1979. 254. 11264-1 1271. 1241 Gordon. J . S., Rosenfeld, B. I., Kaufman, R. and Williams. D. L., Biochemistry 1980. 19.4395-4402. [251 Shastri, K., Isackson, P. J., Fishback, J . L., Land, M. D. and Reeck, G. R., Nucleic Acids Res. 1982,10, 5059-5072. [261 Chiva, M. and Mezquita. C., FEBS Lett. 1983, 162. 324-328. 1271 Rabbani, A,, Goodwin, G. H. and Johns, E. W.. Biochem. J. 1978, 173,497-505.
Alternative methods for fixing and staining gliadins in polyacrylamide gels Staining efficiencies ofCoomassieBrilliant Blue G-250(CBB G-250) and Coomassie Brilliant Blue R-250 (CBB R-250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R-250 in 12 % TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low- and highmobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G-250 and R-250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G-250 in 2 % TCA, in 2 % T C A containing 5 % sodium sulfate, and in 2 % and 5 % phosphoric acid containing 5 % sodium chloride or 5 % sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R-250 in 12 % TCA.
1 Introduction Polyacrylamide gel electrophoresis (PAGE) of gliadins (“gliadin fingerprinting”) has become an invaluable technique for identifying wheat genotypes and for establishing homogeneity. Although extraction procedures and electrophoretic conditions vary considerably among laboratories, staining procedures employing Coomassie Brilliant Blue R-250 (CBB R-250) are used almost universally, and use of CBB R-250 in 12 % trichloroacetic acid (TCA) has become virtually standard [ 1-41. Some procedures also call for prefixation and/or washing in 12 % T C A [4]. Employment of CBB R-250 with T C A as fixative and dye carrier for gliadin P A G E is a natural consequence of successful application of the procedure to Correspondence: Dr. Robert L. Clements, USDA-ARS Soft Wheat Quality Laboratory, Wooster, OH 44691, USA Abbreviations: CBB, Coomassie Brilliant Blue; PAGE, polyacrylamide gel electrophoresis; TCA, trichloroacetic acid Mention of firm name or product does not constitute endorsement by the U S . Department of Agriculture. 0 VCH Veriagsgeseilschaft mbH, D-6940 Weinheim, 1990
other proteins. This procedure takes advantage of the effectiveness of T C A as a universal protein precipitant. The introduction of high concentrations of T C A (12.5 %)was a result of efforts to optimize fixation and staining with minimum background [ 5 , 61. Under these conditions the dye is in a colloidal state, and although capable of binding to protein, it is essentially insoluble in the medium; background therefore is negligible. The use of Coomassie dyes for staining proteins in gels in T C A and other media has been summarized in several reports [7-91. CBB R-250/12 % T C A also provides good results as a gliadin fixative and stain. However, the gliadins, as prolamines, represent a class of proteins that exhibit unique solubility properties. They are essentially insoluble in aqueous buffers and salt solutions that disperse albumins and globulins. On the other hand, the gliadins are soluble in alcohols and weak acids, agents that precipitate and/or denature many proteins. The CBB R-250112 % T C A procedure has been used extensively in this laboratory for routine fixation and staining of gliadins separated by P A G E [ 10-121. However, with continued use of the technique, several drawbacks have become 01 73-0835/90/0202-0123%2.50/0
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Electrophoresis 1990,11, 123-128
evident: TCA is relatively expensive:, and as used in this laboratory (1 2 % T C A for fix/stain only, and not for prefixation or washing) has accounted for about 75 % of the cost of reagents used for PAGE. Also, T C A is extremely corrosive and caustic, and the large quantities required for routine use in 12 % solutions pose a risk to personnel and equipment. Because aqueous concentrated T C A decomposes on standing, 12 % solutions must be prepared just before use. Moreover, decomposition products of TCA include chloroform, creating a potential disposal problem.
However, proteins employed in the study were albumins and globulins. Also, their recommended procedures all included prefixation in 12 % TCA. Several additives (e. g., salts) were included in evaluations of various media. The authors concluded that best staining occurs when conditions are adjusted so that the dye is in a colloidal form which does not precipitate. The unique behavior of the gliadins suggests that nonconventional approaches might be employed for fixation. The studies by Neuhoff et al. (91 show that the Coomassie dyes can function in a wide range of media if conditions result in the desired colloidal state. The objectives of the following study were to investigate alternative media for fixation and staining of gliadins in polyacrylamide gels, and to determine suitability of the media as carriers for CBB G-250 and CBB G-250dyes. A specific objective was to reduce or eliminate requirements for T C A in the media.
Recently, Neuhoff et al. [91 reported results of a systematic and detailed study of protein staining in polyacrylamide gels with R-250 and G-250 dyes. The authors included several media in their study, and concluded best results were obtained with CBB G-250 in TCA, perchloric acid or phosphoric acid.
Table 1.Effects ofstaininggliadinsinpolyacrylamidegel using CBB G-250 and R-250in variousmedia ~
Medium Acidic 5 % Acetic acid 5 % Acetic acid 5 % Acetic acid 10 % Acetic acid 1.2 % Picric acid (satd.) 1.2 % Picric acid (satd.) 1.2 YOPicric acid (satd.) 1.2 % Picric acid (satd.) 2 % Phosphoric acid 2 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 5 % Phosphoric acid 2 % TCA 2 % TCA 2 % TCA 2 % TCA 2 %TCA 2 %TCA 5 % TCA 12 % TCA Basic 5 % Sodium bicarbonate 5 % Sodium bicarbonate 5 Yo Sodium bicarbonate 10 % Sodium bicarbonate 10 % Sodium carbonate 10 % Sodium carbonate 10 % Sodium carbonate Neutral Water, deionized Water, deionized Water, tap Water, tap 5 % Sodium chloride 5 % Sodium sulfate 0.1 M Sodium picrate 0.1 M Sodium picrate
Salt
5 % NaCl 5 % Na,SO, 10 % NaCl 5 % NaCl 10 % NaCl 5 % NaCl 5 % NaCl
5 % NaCl 5 % NaCl 5 % Na,SO,
5 % NaCl 10 % NaCl 10 % NaCl 5 % Na,SO, 5 % NaCl
10 YONaCl
5 YONaCl 10 % NaCl
~
~~~~
a) Dye
b) Intensity
C) Background
d) Slow bands
e) Fast bands
G G G G G R G G G R G R G R G G R G G R G G R
3 2 2 2 2 2 3 2 3
2 3 2 3 2 2 2 2 3 3 3
2 3 3 3 2 2 3 3 3
2 1 2 2 3 3 3 3 3 3 2 2 3 3 3 3 3 2
1
3 3 3 1
3 3 2
3 3 1
3 3 3
G R G G G G G
2
G R G R G G G R
2 2 3 3 2 2
1
2 2 3 1 1
1 1
2 3 3 3 3 3 3 3 3 3 3 3
1
2 2 3 2 3 3 3 3 3 2 3 3 3
2 2 2 2 2 3 3
2 2 3 3 3
2
1 2 3 2 3 3 2 2
3 3 3 3
2 2 1
1 1
a) G, CBB G-250; R, CBB R-250 b) Overall intensity of stain on scale of 1 (very weak) to 3 (intense) c) Background on scale of 1 (heavy) to 3 (negligible) d) Fixation and intensity of slow bands on scale of 1 (very weak) to 3 (all fixed and fully stained) e) Fixation of fast bands on scale of 1 (very diffuse) to 3 (sharp)
1
2 3 2 3 1
1 1
2 2 1 1 2 2 2 2 2 2 2 1
Electrophoresis 1 9 9 0 , I I , 123-128
2 Materials and methods 2.1 Chemicals Acrylamide was “suitable for electrophoresis” grade from Sigma (St. Louis, MO). Dyes were CBB G-250 from J. T. Baker Chemical Co. and CBB R-250 (Serva Blue R) from Serva Fine Biochemicals (Westbury, NY). Other reagents were reagent grade chemicals from various sources. None of the reagents were further purified. Unless otherwise noted, water was double-deionized with a specific conductivity ofless than 0.3 mS/cm. T a p water, where specified, had a specific conductivity of about 700 mS/cm.
2.2 Electrophoresis The electrophoresis procedure, employing 16 x 18 cm x 1.5 mm gels containing 12 % total acrylamide with 3 % crosslinkage (12 % T, 3 % C ) and buffered with acetic acid, was described previously L 121. Wheat meals were extracted with ethylene glycol(2 mLper g meal) [ 101andextracts were stored at -20 OC. The same set of extracts was applied repeatedly to gels to provide replicate gels for staining studies. Loads were 8 pL per well on 10-well gels.
2.3 Staining and washing
Fixing and staining of gliadins
125
staining may have contributed to the loss of sharpness and resolution. Because of the effects in these two regions, media were evaluated for “slow band” and “fast band” staining, in addition to overall stain intensity and background. These four attributes were judged on a scale of 1 (poor) to 3 (excellent), with CBB R-250 in 12 % T C A serving as a standard(Tab1e 1). A dye-medium system with a combined score of 12 was thus judged to be as good as the standard. Although subjective, a score provides an indication of the potential usefulness of a particular system. However, the scores do not take into account such attributes as time required for staining or destaining, or stability of the stain. As noted earlier, staining efficiencies of CBB G-250 and CBB R-250 are a function of the colloidal state of the dye [7-91. Generally, acidic systems are employed for protein fixation and staining, and in such systems colloidal state is determined by strength and concentration of the acid and by the nature and concentration of any added electrolytes [9]. Optimum conditions are not the same for CBB G-250 and CBB R-250, however. Also, conditions that are optimum for staining may not provide fixation of all proteins. Experiments with simple acid systems showed that CBB R-250 in 5 %phosphoric acid resulted in excellent overall stain and low background, but gave weak slow bands and diffuse fast bands (Fig. lc). Saturated picric acid gave similar results, but with higher background (Fig. Id). Staining with CBB G-250 or CBB R250 in 5 % or 10 % acetic acid (not shown) resulted in poor fix-
Upon completion of electrophoresis, gels were transferred immediately to the fixative (400 mL). Dye (3 mL 0.5 % in 50 % aqueous ethanol) was then added dropwise with agitation. Additional dye was added in 18-20 h if concentration appeared to be limiting. Gels were agitated periodically during staining and were stained for different periods, depending upon the rate of staining. When stain intensity appeared to have attained a stable level (2-4 days), staining solution was decanted and the gel was washed. In most instances, avery short rinse (< 1min) with aqueous 50 % methanol110 % acetic acid followed by a second short rinse with aqueous 7 % acetic acid15 % methanol sufficed. Gels stained with CBB R-250 (and a few gels stained with CBB G-250) required slightly more washing, but no gels were destained other than to remove residual medium and precipitated dye. Gels were photographed immediately after washing unless otherwise noted. (The two outer lanes on each gel were deleted from the photographs). Results were evaluated by visual comparison of stained gels with gels stained with CBB R-250 in 12 % TCA.
3 Results and discussion 3.1 Comparison of different staining conditions Comparisons of gels stained under the various conditions showed qualitative differences, as well as differences in overall stain intensity and background, i. e., all bands were not affected in the same way. The low-mobility gliadins or ogliadins [ 131, in particular, were differentially fixed and/or stained. Some bands in this region were extremely weak or lacking in many gels in which the more mobile gliadins appeared to be fully fixed and stained. Also, those gliadins of moderate to high mobilities (including the a- and P-gliadins) showed differential effects, evident as diffused bands. Although appearing to be caused by incomplete fixation, poor
Figure 1 . Gliadins stained in 12 Yo polyacrylamide gels with Coomassie Brilliant Blue R-250 in (a) 12 % TCA, (b) 2 o/o TCA (c) 5 % phosphoric acid, and (d) saturated picric acid. Cultivars (left to right, all gels): Logan, Caldwell, Cardinal, Compton, Hart, Pike, Titan, Tyler.
126
R. L. Clements
Electrophoresis 1990, 11, 123-128
Figure 2. Gliadins stained in 12 % polyacrylamide gels with Coomassie Brilliant Blue G-250 in acidic media: Effects of adding sodium chloride and sodium sulfate (vertical axis) to 5 % acetic acid, 5 % phosphoric acid and 2 Yo TCA (horizontal axis). Cultivars (left t o right, all gels): Logan, Caldwell, Cardinal, Compton, Hart, Pike, Titan, Tyler.
ation and staining. On the other hand, CBB R-250 in 2 % T C A (Fig. lb) gave results comparable to those obtained with CBB G-250 in 12 % TCA (Fig. 1a), with no apparent deficiencies in fixation.
but resulted in some diffusion of the fast bands, particularly in 2 % TCA. Incorporation of 5 % sodium sulfate in the three acid systems provided good fixation and staining of all bands. Similar results were obtained with saturated picric acid (not shown).
3.2 Addition of electrolytes
Experiments with neutral and basic salt solutions indicated gliadins are fixed in such media (as evidenced by turbid bands), but the conditions may not promote staining. CBB G-250 provided good staining under alkaline conditions (5 %sodium bicarbonate and 5 % sodium carbonate in Fig. 3a and b). However, CBB G-250 in 5 % sodium chloride or 5 % sodium sulfate produced very weak stains (Fig. 3c and d). Stephano etal. [14] have suggested sodium picrate (pH 7.0) as a medium for staining polypeptides in gels. However, neither CBB G-250 nor CBB R-250 provided satisfactory staining of the gliandins in this medium (Table 1).
Addition of electrolytes to acidic systems not only influences the colloidal state of the dye, but also affects fixation of gliadins. Fig. 2 shows effects of adding 5 ?41 sodiumchlorideor 5 % sodium sulfate to 5 % acetic acid, 5 % phosphoric acid and 2 % TCA, with CBB G-250 as the dye. Although the slow bands appeared to be fully fixed and stained in 2 % T C A without added salt, many of these bands were weakly stained in gels in 5 % acetic acid or 5 % phosphoric acid. Addition of 5 % sodium chloride greatly improved staining ofthese bands,
Electrophoresis 1990,11, 123-128
Figure 3. Gliadins stained in 12 Yo polyacrylamide gels with CBB G-250 in basic and neutral salt solutions: (a) 10 % sodium bicarbonate. (b) 10 % sodium carbonate, (c) 5 YOsodium chloride, and (d) 5 % sodium sulfate. Cultivars (left to right, allgels): Logan, Caldwell, Cardinal. Compton. Hart, Pike, Titan, Tyler.
3.3 Staining in water The results suggested the gliadins can be fixed and stained under a wide range of conditions, including relatively mild conditions. Therefore, experiments were performed using water alone as amedium. Goodresults were obtained with both CBB G-250 and CBB R-250 in tap water (Fig. 4), but qualitative differences were noted in patterns from the two dyes. In gels stained with CBB G-250, the slow bands were fixed and stained, but the fast bands were moderately diffused; in gels stained with CBB R-250, the slow bands were only partially stained but the fast bands were relatively sharp and intensely stained. In deionized water, CBB G-250 gave moderately stained patterns in which the slow bands were almost imperceptible. CBB R-250 produced relatively strong patterns, but as in gels stained with tap water, several slow bands were lacking. The behavior of the dyes in deionized water suggests the dyes alone may be capable of fixing (i. e., precipitating) some gliadins. Of the four water systems, only CBB G-250 in tap water resulted in staining of the slow-moving doublet noted in all eight cultivars.
3.4 Other staining systems Several systems in addition to those summarized in Table 1 were studied. Those described were included to illustrate
Fixing and staining of gliadins
127
Figure 4. Gliadins stained in 12 % polyacrylamide gels with CBB G-250 (left) and CBB R-250 (right) in tap water (top) and deionized water (bottom). Cultivars (left to right, all gels): Logan, Caldwell, Cardinal. Compton, Hart. Pike, Titan, Tyler.
systems that appear to be potentially useful, as well as to show quantitative and qualitative effects of various media on fixation and staining. Among media studied were ammonium sulfate solutions and acidic systems containing ammonium sulfate, including the 2 % phosphoric acid/6 % ammonium sulfate system of Neuhoff et al. 191. Results from these media were similar to those obtained with sodium chloride as salt, i. e., gels exhibited some degree of diffusion of the fast bands. The results suggest many other systems could provide acceptable fixation and staining of the gliadins. Requirements for fixation appear to be minimal, permitting adjustment of conditions to provide optimum staining. Several systems were judged to be comparable to the standard CBB R-250 in 12 % TCA. Among these were CBB G-250 in 2 % TCA, 2 % TCA containing 5 % sodium sulfate, and 2 % and 5 % phosphoric acid containing 5 % sodium chloride or 5 %sodium sulfate. In addition to reducing or eliminating TCA, these systems offer other advantages. CBB G-250, in these media, provides a highly transparent solution that permits easy observation during staining. Background is negligible, and no destaining is required (other than rinsing to remove precipitated dye). Stability of stain, as such, was not studied, but patterns appeared to be as stable as those obtained with CBB R-250 in 12 % TCA. The current method of choice in this laboratory is CBB G-250 in 2 % TCA/5 %sodium sulfate. Although the sodium sulfate is not essential, the salt appears to promote sharper bands and cleaner, stronger gels.
128
P. Masson et al.
Electrophoresis 1990,11, 128-133
The system has been applied routinely to more than 100 gels with excellent results. Gels may be allowed to remain in the staining solution for extended periods without deterioration of patterns. Stained gels wrapped in polyethylene film and stored for several months at 4 "C retained strong patterns. Storage for shorter periods in 5 % sodium sulfate at room temperature has also given good results.
4 Concluding remarks Staining gliadins with CBB R-250 in 12 c% TCA has become universal, primarily because the method provides good results. However, under some circumstances, it may be desirable or necessary to reduce or eliminate requirements for TCA. It appears that requirements for gliadin fixation are minimal, and that concentrated TCA is necessary only to provide suitable conditions for the staining process. Substitution of CBB G-250 for CBB R-250 permits staining in media other than 12 % TCA, and can provide results comparable to those obtained with CBB R-250 in 12 % TCA. Evaluation of results, together with comparisons of cost, convenience and other attributes may show that an alternate procedure offers specific advantages without sacrificing staining el'ficiency.
5 References [ 1I Autran, J. C., Bushuk, W., Wrigley, C. W. andzillman. R. R.. Cereal
Foods World 1979,24,471-472. 121 Cooke, R. J., Electrophoresis 1984,5,59-72. 131 Lookhart, G. L., Jones, B. L., Hall, S. B. and Finney. K. F.. Cereal Chem. 1982,59, 178-181. 141 Wrigley, C. W., Autran, J. C . and Bushuk, W., Adv. Cereal Sci. Technol. 1982,5,211-259. IS1 Chrambach, A., Reisfeld, R. A., Wyckhoff, M. and Zaccari.J..Anal. Biochem. 1967,20,150-154. 161 Rodbach, D. and Chrambach, A.,Anal.Biochem. 197 1.40.95-1 34. [71 Wilson, C. M., Methods Enzymol. 1983,91, 263-278. 181 Reisner, A. H., Methods Enzymol. 1984,104,439-441. [91 Neuhoff, V., Stamm, R. and Eibl, H., Electrophoresis 1985, 6. 427-448. [ 101 Clements, R. L., Cereal Chem. 1987,64,442-448. [ 11 I Clements, R. L., Cereal Chem. 1988,65, 150-152. I121 Clements, R. L., Electrophoresis 1988,9,90-93 1131 Kasarda, D. D., Bernardin, J. E. and Nimmo, C. C..Adv. Cereal. Sci. Technol. 1976,1, 158-236. 1141 Stephano, J. L., Gould, M. and Rojas-Galicia, L., Anal. Biochem. 1986,I52,308-313.
Received July 28, 1989
Patrick Masson' David M. Arciero2 Alan B. Hooper2 Claude Balny3 'Centre de Recherches du Service de Santedes Armees, Unite de Biochimie, La Tronche *Departmentof Genetics and Cell Biology, University of Minnesota, Saint Paul, MN 31nstitut National de la Sand et de la Recherche MBdicale, Unit6 128, B.P. 505 1, Montpellier
Electrophoresis at elevated hydrostatic pressure of the imultiheme hydroxylamine oxidoreductase The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 2 5 "C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.
1 Introduction Hydroxylamine oxidoreductase (HAO; EC 1.7.3.4) is a multiheme protein from the aerobic nitrifying bacterium Nitrosomonas [ 11 which catalyzes the oxidation of hydroxylamine to nitrite. H A 0 is a multimer of an a-subunit (- 63 kDa) containing at least 6 c-hemes and one active site which contains a
Correspondence: Dr. Patrick Masson, CRSSA, Unite de Biochimie, B.P. 87, F-38702 La Tronche Cedex, France Abbreviations: BSA, bovine serum albumin; HAO, hydroxylamine oxidoreductase; SDS, sodium dodecyl sulfate 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
heme-like structure, P-460 [2,3]. H A 0 can be isolated with a second 11 kDa c-cytochrome (referred to as a "P-subunit") which is not necessary for activity and is not present in current preparations of the pure enzyme 141.Thenumber of asubunits in the enzyme is not known. The molecular size, approximately 180-200 kDa [3, 51 based on hydrodynamic measurements, is uncertain since the shape of the protein is unknown. Treatment of H A 0 with sodium dodecyl sulfate (SDS) and then SDS/mercaptoethanol results in bands of progressively higher apparent molecular weight (125, 195 and 225 kDa) as seen on SDS-polyacrylamide gels [ 3 ] .The 63 kDa monomer has thus far been observable only after chemical removal of all c heme. This behavior in chemical denaturants has suggested polymerization and/or denaturation of the H A 0 monomer. 0 173-0835/90/0202-0128 $2.50/0
