Buffe:r; extracts of pollen from Secale cereale as used ii+ therapy of pollinosis were prepa~edl. All the extracts were found active upon intradermal test in pollen sensitive subjects. Electro;phoretic and chromatographic analysis revealed that the extracts contain seven ninhydrin reacting components. Two amino acids, proline and [ysine, and five others (very probably polypeptides) were found to be components of the extract. Two major peptide components (C2 and D) were isolated and amino acids composition of acid hydrolyzates determined. Component C2 contained Asp., Glu., Ser., Gly., Thr., Lys., Tyr., (Cys.?), and D component Asp., Glu., Ser., (Cys.?), Gly., Thr., Tyr., V,al., Pro. I:n the original pollen extract\u27s hydrolyzate His., Phen., Leu., not certainly Tryp,, and Isoleu. were found in addition to the amino acids cited above. Of carbohydrates glucose, galactose and fructose were detected

N. Kohler-Kubelka

N. Muić

English

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C R 0 AT I C A C H E M I C A A C T A 31 (1959) 133 CCA-159 577 .1.004.12:541.41 :616-056.3 :581.16.082.2:613.14 A Contribution to the Biochemical Characterization of Allergens in Pollen Extracts of Secale Cereale N. Muic and N. Kohler-Kubelka Department of Applied Biochemistry, »Andrija Stampar« School of Public Health, Faculty of Medicine, University of Zagreb and Institute for the Production of Sera and Vaccines, Zagreb, Croatia, Yugoslavia Received November 5, 1959 Buffe:r; extracts of pollen from Secale cereale as used ii+ the-rapy of pollinosis were prepa~edl. All the extracts were found active upon intradermal test in pollen sensitive subjects. Electro;phoretic and chromatographic analysis revealed that the extracts contain seven ninhydrin reacting components. Two amino acids, proline and [ysine, and five others (very probably polypeptides) were found to be components of the extract. Two major peptide compo-nents (C2 and D) were isolated and amino acids composition of acid hydrolyzates determined. Component C2 contained Asp., Glu., Ser., Gly., Thr., Lys., Tyr., (Cys.?), and D component Asp., Glu., Ser., (Cys.?), Gly., Thr., Tyr., V,al., Pro. I:n the original pollen extract's hydrolyzate His., Phen., Leu., not certainly Tryp,, and Isoleu. were found in addition to the amino acids cited above. Of carbo-hydrates glucose, galactose and fructose were detected. INTRODUCTION In spite of great amount of experimental research on the chemical nature of pollen allergens_in the prast fifty yeairs, there is no definite answer yet which group of chemical compounds from pollen extracts possesses the potency of allergizing. The most favourable point of view is that the proteins from pollen cells are responsible for its aillergenic capacity. Formerly Prausnitz1 had published that the only albumin portion from fractionated rye pollen proteins has showed the allergenic properties. Urbach2•3 later on concluded in accordance with the results of his experimental inve,stigati.ons that the allergenic principle appeaired to be associated with globulin and not albumin fractions in the pollen extracts of Secale cereale. In general many investigations' have recently estab1ished the fact again that the pollen allergens are proteinic in nature (Unger and co-w'9rkers4, Roth and Nelson5, Newel6, Cohen et al.7, Abramson, More and Gettner8, Hampton, Johnson Mary and Frankel9). For the most part these studies are concerned about various ragweed al1ergens. Timoty pollen allergellJS have been studied by Ahmmson, Engel and Moore10, a preliminairy report on fractionation of grass pollen has been published by Augustin11, and Herbertson et al. have fovestigated allergens from alder pollen12. Because of va:I'liable success in the desensitation therapy of pollinosis with pollen extracts, prepared according to the conv;entiooal buffer elution method, 134 N . MUIC AND N . KOHLER-KUBELKA we ,supposed thait native allergens occurring in pollen could be chemically changed during extraction procedures. The present communication is an at-tempt to acquire some eviden.oe about the nature of pollen allergens from Secale cereale pollen in buffer extracts. EXPERIMENTAL Polle?\ For the preparation of pol!len extracts of Secale cereale, pollens were collected. during summer 1958 in the r egion of Zagreb, Cr~oatia, Yugoslavia. The pollen materia'1 was dried in a dessicatoc and was kept cool and dry. · Extraction The preparation of buffer extract from pollen material was .accomplished in such a manner that the crude material underwent a1s little chemical alteration as possible. The defatting of pol!len was carried out by washing it with successive portions of diethylether (;pro narcosi). The ether was removed from defatted material merely by allowing it to evaporarte in the air at room tempera.ture. Buffer saline, as used at the University laboiratories of Michigan, was th.e· extracting medium: NaCl 5 g.; KH2P04 0.36 g.; Na2HP04 7 g.; Phenol 4 g.; distilled water to makie 1'000 ml. Final pH of the buffer was clo,se to 8. Adjustement was made using NaOH or HCl as needed. · Extracts of Secaie cereale polllen were prepaired by adding 1 g. of ether-defatted material to 49· ml. of extracting buffer. After extmcUion a·t room temperature (2.iOC) for 72 hours the extracts were filtered under sterile condition through a Seitz filter, aliquots (5 ml.) were rapidly frozen in ampoolles and lyophil:ized in order to preserve them sterile. The total nLtrog~n content, determined hy micm Kjeldahl method was 0.172 mg. per ml. of original extracts (1 g. of pollen in 4·9 ml. of buffer). Biofogical investigations of buffer extracts from pollen of Secaie cereale were peiformed by Dr. M. Mimica on pl)llen sensitive persons, by intradermal and scratch test art the Department of Medicine, University of Zagreb. P<11per chromatography Both descending chromatographyta,14 for solvents phenol-water (4:1). and ascending chromatogmphyls for n-buta:nol-acetic acid-water (4:1 :1 and 4:1 :5) were· used for · sepa.r:ation of pollen peptides a:nd amino acids in hydrolyzates. In most experiments 0.02 ml. of cone. extract (-0.1 g. of pollen in 0.2 ml. of water) or an adequate amount of hydro1yzed extract was applied on Whatman No. 1 paper strip. The buffer extraets were desalted by diaJys~s against distilled water (48h, 20), the hydrolysates by elecitrodialysis in Consden apparatus. For isolation of peptide fractions, the concentrated extract (0.06 ml.) was applied on a wider (11 cm.) strip of filter paper and developed one-dimensionally using phenol-water (4:1} as eluent. The position of spots on air dried chromatograms were detected by employing minute dmps of etha:nolic ninhydrin · along the paper strip. Separated fractions were cut out from paper chromafogram and washed out from fhlt~r paper with warm water and hydrochloric acid (20°/o)<. Isolated fractions were hydrolyzed with 200/o HCl in the sealed glass tubes for 24 hours at 1050C. The acid hydrolyzates were evaporated at 1oooc and dried in vacuum desiccator over sodium hydroxide and cafoium chloride. The amino acids were detected by spraying of the developed paper chromato-grams with 0.30/o ninhydrin in ethanol. For the identification of .a mino· acids a guide· chromatogram with corresponding artificial amino acid mixture was simultaneously developed. · Paper electrop~oresis Zone electrophnresis was carrried out according to the method published by Cremer and Tiselius16, and Grassm.ann, Ha nnig and Knede111. Veronal-veronal sodium buffer pH 8.6, ionic strength 0.05 ; . Whatman No. 1, 5-10 V/cm ; 1-Q mA; 4 hours, 0.02 ml. of cone. extract of Secale cereaie. The proteins were fixed by drying at l050C for 10 minutes and stained with 0.030/o tetrabromopheno'1blue in 10/o aqueous. THE BIOCHEMICAL CHARACTERIZATION OF ALLERGENS 135 mercurichloridets, the peptides by spraying the eleotropherograrrn with 0.3~/o ethanolic ninhydrin. Photometric evaluation When dried the .e.Jectropherograms were made translucent by immersing them into a mixture of panaffin oil and Ceoo.x (Behring Werke, Ba~er) in petroleum ether. The evaluation of transmittance of electropherograims and chromatograms was carried out in 1 cm. wide bands at intervals of 1 mm., with a modified Fischer electrophotorneter and a green filter No. 5251s. RESULTS AND DISCUSSI ON All buffer extra.cts, made :firom pollen of Secale cereale and diluted with saline, weire found to be sk1n I'eactive in high dilution upon intradermM test in Secale cereale pollen sens1itive subjects. These extracts did not show some of the character.istic reactions for true proteins (mol. weight over 10 000). Concentrated pollen extract iia.iled to precipitate with trichlomcetic acid and coagulate by boiling. The components from extraicts could not be fixed on filter paper in chromatographic procedures by heating them at 105°C, nor precipi-tated with mercuric chloride solution. When chromatograiphed, these com-ponents could be well stained on filter paper with bvomopheno•lblue but they were badly fixed a.nd could be washed out by water18• These results are in accord with prior experience of W. P. Dunbar with poUen extracts, as cited by Urbach2 and Hansen19. Such properties are common with proteins having molecular weight less than 10 000. In fact Abramson8 and Newel20 and Augu-stin11•21 have succeeded in isolating the biologically active fractions from ragweed and grass pollen extracts, with molecular weights between 5 OOO and 13 OOO re.spectively. E d mm Fig. 1. Electropherogram with superimposed photometric evaluation. 0.02 ml. Secale cerea!c cone. pollen extract; veronal-veronal sodium buffer pH 8.6, ionic strength 0.05: 2.6 V/cm, 1.8 m l., 4 hours, 0.3'/• ninhydrin in ethanol, Whatman No. 1. 13,5 N. MUIC AND N. KOHLER-KUBELKA Electrophoretic separation of concentrated extract of Secale cereale pollen in alkaline (pH 8.6) veronal-veronal sodium buffer of satisfactory ionic strength (0.05) and potential gradient (8.6 V/cm.) was achieved (Fig. 1). The rye allergens were separated into three groups with at leaist seven components. Two com-ponents, a and b, migrated at 'PH 8.6 towards the anode not quite distinctly resolved, and four components migrated towards the cathode. The mino1r <::ornponent c did not migrate at these experimental conclitiorns. Because' we cannot assume that a stoichiometric relationship exists between the denaturated proteins and the adsorbed dye, the photometric evaluation has only relative v;alue of qualitative analys1s. None of the components separated could be well fixed on paper fibres by heating; consequently the peptide components dyed with bromophenol blue, were wa1shed out during the staining procedure.18 Since the electropherograms were stained with ethianolic ninhydrin, we could not differentiate peptides from ~mino acids or exclude the overlapping of peptides and amino acids bands. Paper chromatography was performed with v;arious eluents. The appli-cation of consecutive elution22 of the components from the paper, with salt solution in progressive concentriations, has not giiven satisfactory result!?. -Better separation of pollen peptides was achieved using n-butanol-acetic acid-water solvents. E Fig. 2. Chromatogram with superimposed photometric evaluation; 0.015 ml. Secate cerea!e cone. pollen extract one-dimensionally developed, solvent phenol-water (4:1); 0.3'/o ninhydrin in ethanol. S =start, F =front. The relative distances (Rf) traveled by components were not however great enough for complete resolution of mixture. Spraying of the developed THE BIOCHEMICAL CHARACTERIZATION OF ALLERGENS 137 chromatogram with ninhydrin revealed 5-6 polypeptide bands, from which two major bands could be well distinguished beciause of fairly different migra--t1on rate (Rf 0.14 and 0.31). The third component reacted with ninhydrin producing yellow colour and with sodium salt of 1,2-naphthoquinone-4-sulphonic acid, red colour. This: component, which showed the same migration rates on paper in all eluents as proline, should be considered as identical with it. The chromatograms developed one-dimensionally with aqueous phenoI revealed seven well separated components. Photometric evaluations of these chvomatograms are presented in F1ig. 2. The groups of components represented as peaks in this diagram cannot be identified with those in Fig. 1 because of a different distribution of the components during electrophoresis. 0 c: ti .c: c. n-Butanol-acet.acid . () 1 E ~I/ 2. E, ·······-0.07 ---·- --0.14 -----. -o. 26 ------. o.so -----.. o. 52. t D ---·· ··0.78 - . --- --0. 89 - - - -·. -0.96 Fig. 3. Two-dimensional chromatogram of pollen extract (0 .03 ml.) from Secate cereate. First direction, ascending: n-butanol-acetic acid-water (4:1:5); second direction, descending : phenol-water (4:1). il38 N. MUIC AND N. KOHLER-KUBELKA Moat successful was two-dimensional chromatography of concentrated buffer extract from Secale cereale pollen. These chromatograms were deve-loped in one dimension with butanol-acetic acid-water and in other with aqueous phenol. Fig. 3 shows the positionis of components rea;cting with nin-hydrin, on a two-dimensionally developed chrom,atog,ram. At least eight com-ponents could be well distinguished oailled A, B1 , B2, C1 , C2, D, E1 and E 2 • The Rf values corresponding to the components are added in the drawing. The components ·B 1 and B2 aire weakly separated and badly dyed with ninhydrin. It is evident from the driawing that the chief bands C2 and D have satisfactory different relative mignaitiori rates (Rf values) in phenol-water solvent to be successfuly sepa rated on one-dimensiona;lly developed chromatogram. Ac-cording to the results of the amino acids analy;sis component C1 proved to be identical with lysine and E1 witl:i proliine. All components iin Fig. 3 denoted with capitals (A, B 1, B 2, C11 C2, D,, E1, E 2), correspond to those in Fig. 2. For the preparation of components C2 and D one-dimensional chromato-grams with aqueous phenol were used. Larger sheets of paper (11 cm.) were streaked with 0.06 ml. of concentrated buffer extract from Secale cereale pollen (0.5 g. pollen per ml.) across the paper strip, thus a narrow and uniform band (0.3 cm./10 cm.) of substance was obtained. This sheet was then developed in the same manner as previously described, and the components from the chromatogram isolated. After isolation the substances C2 and D were hydro-lyzed and the amino acids composition of hydrolyzates accomplished by pe,r-forming two-dimensiona:l chromatograms. According to our results the com-ponent C2 contaiins amino acids as fo1lows: .Asp., Glu., Ser., Gly., Thr., Lys., Tyr., (Cys.?) and D component Asp. , Glu., Ser., (Cys.?), Gly., Thr., Tyr. , Val., Pro. In the acid hydrolyzate of the ori~inarl pollen extract were found all amino acids cited above and some other more: His., Phen., Leu., (Isoleu.?), and in the alkaline hydrolyzate not certainly tryptophan. For detection of carbohydrates concentrated pollen extract was chromato-grap_hed with phenol-water as solvent. Spraying of chromatograms with anilin-phosphate23 revealed three spots with migration rates corresponding to glucose, fructo·se and gaJactose. Similar results have been found by Augustin24 in grass pollen and Hampton et al.9 in ragweed pollen extracts. From the results of our investigation it is evident that the favourable group of protein derivatives in buffer extracts from pollen of Secale cereale are polypeptides. Since it is known that pollen proteins show relatively high resistan.ce to digestive enzymes1•25 we can assume that nativ;e enzymes from pollen are responsible for the splitting of the proteins :in rye pollen e-xtract. The fractions prepared chromatographically with butanol-acetic acid or phenol could not be used for skin test on sensitive subjects. Results of biofogical testing of suitable isolated fractions from Secale cereale pollen allergens will be published elsewhere at a later date. REFERENCES 1. 0. Prausnitz, in W . Kolle, R. Kraus, and P . Uhlenhuth, Hand. der pathogenen Mikroorganismen, IH. Auf., III, 1 Berlin 1930 p. '133. 2. E. Urbach and P. M. Gott 1 i e b :, AHergy, 2nd ed., New Yo1"k 1946', p. 249. 3 . . E. Urbach, Klin. Wochschr. 12 (19·3·3.) 1797. 4. L. Unger, M. B. Moore, H . W. Cromwel!l, and C. H. Seeber, J. AHergy 5 (1934) H5. THE BIOCHEMICAL CHARACTERIZATION OF ALLERGENS 139 '5. R. R. Roth and T . Ne 1 son, J. Allergy 13 (1942) 283. 6. J. M. New e 11, J . Allergy 14 (1943) 444. 7. M. B . Cohen and H. J . Fri e dman, J. Allergy, 14 (1943) 368. 8. H . A. Abramson, D. H. Moore, and H. H. G ettner, Proc. Soc. Exp. Biol. Med. 46 (,1941) 46. 9. S. Ha m p t .o n, Mary C. J oh n son, and S. Franke 1, J. Allergy 26 (1955) 16. 10. H . A. Abramson, M. G. Engel, and D . H. Moore, J. Allergy 14 (1942) 65. 11. R. Augustin, Biochem. J. 54 (1953) Xa. 12. S . Her ib er t son and J. p·o rat h, Acta Chem. Scand. 12 (1958) 737. 13-. R. Cons den, A. H. Gordon, and A. I. P . Martin, Biochem. J. 38 (1944) 224. 14. C. E. D en t, Biochem. J. 43 (1948) 169. 15. R. J. W i 1 li am s and H. Kirby, Science (New York) 107• (1948) 481. 16. H. D. Cremer and A. Tise 1 i us, Biochem. Z. 320 (1949) 273. 17. W. Grassmann, R. Hannig, and M. Knedel, Deut. med. Wochsch. 76 (1951) 333. 18. M. Pila!ntanida and N. Mu1c, Arch. Biochem. Biophys. 46 (195'3) 110. 19. K. Hansen, Allergie, G. Thieme Verlag, Stuttgart 1957 p. 444. 20. J. M. Newel, J. Allergy 13 (1942) 177. 21. R. Augustin, Bio chem. J. 54 (1953) X B. 22. N. Mui c, A. M en i g a, and M. F 1 e s, Arch. Biochem. Biophys. 77 (1958) 20. 23 . I. L . B r y son and T . I. Mitch e 11, Na tu re (London) 167 (11951) 864. 24. R. Augustin, Biochem. J. 64 (1956) 28 P . . 25. R. Doer, Die Immunitiitsforschung VIII. Springer Verlag, Wien, 73-74. IZVOD Prilog biokemijskoj kairakterizaciji alergena ekstrakta peludi Secale cereale N. Muic i N. Kohler-Kubelka Ekstrakcijom peludi Secale cereale priredeni su alergeni, koji se upotrebljavaju '1.1 teraipiji bolesnika ·osjetljivih na pelud. Ovi ekstrnkti pokazivali su pozitivnu intra-dermaJnu reakciju kod bolesnika osjetljivih n.a pelud Secale cereale. Elektroforetska i kromatografska an.aliza pokaza!la je, da se ekstrakt sastoj.i iz sedam komponenata, koje su reagirale s ninhidrinom. Utvrdeno je, da su dvije komponente aminokiseline, prolin i lizin, a drugih pet su vjernjatno polipeptidi. Dvije vece, kromatografski dobro razdvojene komponente C2 i D, bile su izolirane. U hidrolizatu ovih komponenata odredene su ~romafografski aminokiseline. Komponenta C2 sastoji se iz aminokiselina: asp., glu., ser., gli., treo., liz. , tir., ~cis .? ); a komponenta D iz asp., glu., ser., (cis.?) gli., treo., tir., val., pro. U originalnom ek.straktu peludi nadeni su osim gore spomenutih aminokiselina jos i histidin, fenila-lanin i leucin, te trag;ovi triptofana i izoleucina. U koncentriranom ekstraktu peludi Secale cereale nadeni su krnmatogmfijom ugljikohidrati glukoza, galakfoza i fruktoza. ODJEL ZA PRIMIJENJENU BIOKEMIJU, SKOLA NARODNOG ' ZDRAVLJA »ANDRIJA STAMPAR« MEDICINSKI FAKULTET, SVEUCILISTE U ZAGREBU I INSTITUT ZA PROIZVODNJU SERUMA I VAKCINA ZAGREB Prlmljeno 5. studenoga 1959. 

A Contribution to the Biochemical Characterization of Allergens in Pollen Extracts of Secale Cereale

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