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Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V
Author(s) -
MATTHIESEN F.,
LøWENSTEIN H.
Publication year - 1991
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.1991.tb01661.x
Subject(s) - phleum , allergen , sephadex , size exclusion chromatography , molecular mass , microbiology and biotechnology , chemistry , chromatography , fractionation , pollen , biology , biochemistry , immunology , botany , allergy , enzyme
Summary An allergen from Phleum pratense (timothy) pollen. Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromato‐graphy. Phl p V binds IgE from serum of grass‐sensitized donors as revealed in immunoelectrophoretic techniques and in SDS PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS‐PAGE treatment purified Phl p V is identified as two IgE‐binding components. Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30‐kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47‐kD fraction corresponds to Phl p Va/ Phl p Vb as seen in SDS‐PAGE, while the 25‐kD component presumably corresponds to a degradation product present in whole pollen extract. The NH 2 ‐terminal sequence of Phl p V , corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH 2 terminal sequence of other proteins. The ammo acid composition, revealing 26 mote % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison. Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.