The inhibitors function after heating, exposure to organic solvents and low pH during extractions. Thus they are either very stable or else can easily refold. They would be expected to survive cooking.
Nature of main cross-reacting proteins:
These inhibitors are similar to inhibitors from Amaranth (eg. P80211). IgE in allergic sera to buckwheat cross-react with those from latex (De Maat-Bleeker & Stapel 1998 [39]; Schiffner et al., 2001 [502])
Allergen properties & biological function:
These allergens belong to the family of type I potato serine protease inhibitors. These are induced by wounding and are part of the PR-6 family of plant defence proteins. Because rubber plants are wounded in obtaining latex, these are possible, but not established, latex allergens
Allergen purification:
Bolozersky et al (1995) [23] followed an initial extraction from the seed by either a trypsin-Sepharose column or Sephadex G-75 followed by chromatography on Mono Q HR 5/5 in 20 mM K, Na-phosphate, pH 6.8. Protein was eluted by a 0-100 mM NaCl gradient. BWI-1 was then purified by reverse-phase HPLC on a aquapore RP-300 column using a acetonitryl gradient. Pandya et al. (1996) [139] stirred 250 g batches of flour with 2.5l of 2% (w/v) NaCl for 90 min. This was filtered and centrifuged. The supernatant was heated to 80 deg. C for 10 min. Ammonium sulphate was added to 60% saturation. The ppt. was disolved in water, dialysed and lyophilized. The protein was disolved in 6M urea in 0.1M acetic acid and fractioned by gel filtration (Sephadex G-50 equilibrated in 0.1M acetic acid). Fractions were lyophilized and applied to an inactivated trypsin column at pH 8.1. The inhibitors were eluted at pH 2.1 in 0.1M KCl, desalted and lyophilized. The protein was then chromatographed on DEAE-Sepharose CL-6B equilibrated with 50 mM Tris, pH 8.4, with a 0-0.3M NaCl gradient. Peaks were rerun on a Synchropak AX300 HPLC column in 10 mM Na-Pi buffer and eluted using gradients 0-0.2M and 0,2-0.6M NaCl. Park et al. (1997) [143] extracted defatted flour with 10 mM sodium phosphate, pH 7.5, containing 0.15M NaCl with agitation for 2 hours at 4°C. After centrifigation (20 minutes at 13000g), ammonium sulfate was added to 100% saturation and precipitate was collected after 24 hours by centrifugation. After dialysis against 10 mM sodium phosphate, pH 7.5, and gel filtration on Sephadex G-50, the trypsin inhibitory fractions were collected. These were further purified and separated on a DEAE-cellulose column equilibrated with 10 mM Tris/HCl pH 8.0, with elution by a linear 0-0.3M NaCl gradient, followed by purification on an FPLC Mono-Q column and RP-HPLC on a Wakosil C-18 column.
Other biochemical information:
References (9)
Belozersky M.A., Dunaevsky Y.E., Musolyamov A.X., Egorov T.A.
Complete amino acid sequence of the protease inhibitor from buckwheat seeds.
FEBS Lett. 371:264-266. 1995
PUBMED ID:
7556606
Kondo Y, Urisu A, Wada E, Tsuruta M, Yasaki T, Yamada K, Masuda S & Morita Y
Allergen analysis of buckwheat by the immunoblotting method
Arerugi 42: 142-148 [Japanese] 1993
PUBMED ID:
8507155
Pandya M.J., Smith D.A., Yarwood A., Gilroy J., Richardson M.
Complete amino acid sequences of two trypsin inhibitors from buckwheat seed.
Phytochemistry 43:327-331(1996) 1996
PUBMED ID:
8862028
Park JW, Kang DB, Kim CW, Ko SH, Yum HY, Kim KE & Hong CS
Identification and characterization of the major allergens of buckwheat.
Allergy 55:1035-1041. 2000
PUBMED ID:
11097313
Park SS, Abe K, Kimura M, Urisu A & Yamasaki N
Primary structure and allergenic activity of trypsin inhibitors from the seeds of buckwheat (Fagopyrum esculentum M.) .
FEBS Lett. 400:103-107. 1997
PUBMED ID:
9000522
Yamada K, Urisu A, Morita Y, Kondo Y, Wada E, Komada H, Yamada M, Inagaki Y & Torii S
Immediate hypersensitive reactions to buckwheat ingestion and cross allergenicity between buckwheat and rice antigens in subjects with high levels of IgE antibodies to buckwheat.
Ann Allergy Asthma Immunol 75: 56-61. 1995
PUBMED ID:
7621062
Yano M, Nakamura R, Hayakawa S & Torii S
Purification and properties of allergenic proteins in buckwheat seed.
Agri Biol Chem 53, 2387-2392. 1989
PUBMED ID:
unknown
[241]
This record was last modified on 18-Oct-2006
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