Fractionation of pollen extracts by chromatography. I. Preliminary studies with ragweed extract.

Abstract A method has been presented whereby a minimum of 6 different active components were obtained from ragweed pollen extract. Five of these components readily diffuse through Visking membrane and are therefore of relatively small molecular size. It has been demonstrated that 4 of the diffusible components are peptides and the major amino acid composition of 3 of these peptides has been defined. The fifth diffusible active component is a carbohydrate. In addition to the diffusible active components there is a highly active fraction of ragweed extract which is retained by Visking membrane after 16 days of dialysis against running water. It has been demonstrated that this dialyzed residue contains at least one large polypeptide or protein. The observation that this material is not readily demonstrable by the ninhydrin reaction on paper suggests that the polypeptide or protein contains relatively few free amino groups. The major amino acid composition of the protein or polypeptide components of the dialyzed residue has been determined. The activity of each of the fractions described has been demonstrated by direct intradermal test in ragweed-sensitive subjects and by passive transfer and neutralization techniques. Each ragweed-sensitive subject reacted to the dialyzed residue and a positive passive transfer reaction was obtained with each reaginic serum challenged with this material. Fifteen of 17 ragweed subjects and 22 of 25 reaginic sera reacted to one or more of the diffusible components (CHO, A, B, C, and D). The observations presented (Tables I and II) suggest that each ragweed sensitive subject has an individual pattern of reactivity to the smaller molecularsize components: the data suggesting that most subjects are reactive to certain of these components and not to others, some are reactive to all, and a few to none; however, because of a scarcity of material, observations were made with only a single concentration of the dialysate fractions and, therefore, quantitative variations in reactivity have not been fully evaluated. Regarding the origin of the fractions of dialysate, it is possible that the process of dialysis or the action of lutidin on the extract results in a separation of active fragments from a larger comples antigen. Since the active diffusible components have been identified on chromatograms of whole ragweed extract, the process of dialysis does not seem to be responsible for the breakdown of a large comples. Since electrophoretic and ultracentrifuge studies have indicated the presence of a number of active components of small molecular weight 15 it seems further probable that the active diffusible fractions obtained from chromatograms are not the result of any solvent action on a complex but rather that they are native to ragweed extracts. This study demonstrates that paper chromatography is a valuable adjunct in the investigation of a crude antigenic material and the methods employed might profitably be applied to the study of other crude allergenic extracts. Ragweed extract has been fractionated heretofore by electrophoretic techniques and by more classical chemical procedures. The fractions obtained in those studies have been difficult to evaluate and correlate since their chemical definition has in most studies been based on general qualitative chemical reactions. The methods employed in this study provide not only for a separation of the active components of ragweed extract but also provide extensive information concerning the composition of these components. A continuation of the present study of ragweed extract by paper chromatography, immunologic, and classical chemical methods is in progress.