Comparative Studies on Tree-Pollen Allergens

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Abstract
Gel permeation chromatography of B. verrucosa (BV) crude pollen extract and pooling of the fractions after their fused rocket immunoelectrophoresis antigenic profile allowed the isolation of a purified major allergen designated BV45. This fraction was a glycoprotein with a tentative MW of 10,000 daltons. The estimated MW differed by approximately factor 2 from previously reported values of birch pollen major allergens. This fraction was further purified by polyacrylamide gel chromatography (P-10), high performance liquid chromatography (HPLC) silica-base column and high-volt electrophoresis at different dimensions. The purified proteins revealed an immunochemical identity with the initial material. BV45 and the previously isolated pI 5.18, though different otherwise, demonstrated a reaction of complete antigenic identity illustrated by tandem-crossed immunoelectrophoresis. Both the amino acid and carbohydrate compositions of BV45 were calculated on the basis of the tentative MW obtained. BV45 showed a total carbohydrate concentration of 20% of the dry weight, corresponding to 13-14 residues/mol protein. The amino acid composition obtained reflected structural diversity between the BV45 and pI 5.18. N-terminal analysis followed by HPLC quantitation of the DNA-amino acids for BV5% and the further purification products gave 2 peaks having retention times corresponding to DNS-Cya and DNS-Asp. Fraction BV45 was demonstrated to be a major allergen of birch pollen as assessed by RAST [radioallergosorbent test] inhibition of 7 of 10 reaginic human sera and the specific radiostaining in crossed radioimmunoelectrophoresis of all omit 9 crossed immunoelectrophoresis plates used. RAST inhibition titration of BV45 and BV crude showed qualitative identity of the allergenic determinants of both materials. The question about 1 or several major allergen(s) in birch pollen extract is unsolved as no structural mapping of BV45 and pI 5.18 has been readily available.