The isolation and identification of horse-dandruff allergen

Abstract

The composition of horse-dandruff extract was studied by means of physicochemical and immunochemical techniques, which revealed the presence of at least 7 protein or polypeptide constituents containing varying proportions of conjugated carbohydrate in addition to pigment and dialysable substances. Electrophoretic analyses, both in solution and on paper, provided measures of the relative concentrations of the major protein constituents, which were lettered arbitrarily in order of decreasing mobility in veronal buffer at pH 8.6. Staining of the paper strip patterns with periodic acid-Schiff''s reagent revealed the distribution of protein-bound carbohydrate. Fractional precipitation with saturated ammonium sulfate solution, between limits indicated by preliminary quantitative fractionations, provided a series of precipitates containing varying proportions of the electrophoretic components. All fractions were assayed by prick testing horse-sensitive asthmatics. Allergenic activity was associated with a single carbohydrate-rich constituent (designated "b2"), predominant in the fraction precipitated between 55 and 80% saturation with ammonium sulfate and comprising 64% of the total protein in whole dandruff extract. Additional evidence as to the nature of the allergen was obtained from skin tests with relatively pure constituents isolated by preparative electrophoresis on paper and in a starch column. The precipitin line due to the allergen was identified in antigen patterns obtained by the horizontal gel-diffusion technique and detected in preparations of low skin reactivity, which appeared to be allergen-free according to the less sensitive electrophoretic analysis. A very soluble mucopolysaccharide fraction (containing 48% of hexose), and a crystalline protein (of similar electrophoretic mobility on paper to the allergen, but containing only 0.2% of hexose), were inactive. A highly purified allergen preparation was obtained by the starch electrophoresis of the allergen-rich salt-precipitated fraction. Preliminary chemical analyses showed the allergen to contain 9% of hexose in the form of galactose and mannose, in addition to all the common amino acids. Its molecular weight, determined from osmotic pressure measurements, was 34,000. Evidence is provided that the serological relationship between horse dandruff and horse-serum antigens is probably due to the admixture of serum albumin and globulin with the dandruff constituents.