Separation of Alternaria into protein and carbohydrate fractions with phenyl sepharose.
To evaluate the contribution of high carbohydrate-containing fractions to the allergen content of Alternaria, samples of an Alternaria extract were bound to a phenyl Sepharose column and eluted with Tris buffer containing 4, 2, and 1 mol/L of NaCl and then distilled water. The fractions were dialyzed and lyophilized. The 4 mol/L fraction accounted for 79% of the dry weight and most of the carbohydrate content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions demonstrated significant interfraction differences with the largest number of silver-stained bands in the H2O fraction. Although individual differences in skin reactivity to the fractions were present, geometric means of histamine equivalent pricks were approximately the same with only a 0.5 log dilution higher potency for the 1 mol/L and H2O fractions. The 50% IgE ELISA-inhibition concentration was 100 micrograms/ml for the 1 mol/L and distilled water fractions and 10 micrograms/ml for the other fractions. Since the 4 mol/L fraction accounted for most of the dry weight and had the highest carbohydrate to protein ratio (5.8), purification of Alternaria extracts by removal of carbohydrate fractions may result in significant loss of allergenic material.
Portnoy
J
J
Section of Allergy/Immunology, Children's Mercy Hospital, Kansas City, MO 64108.
Pacheco
F
F
Ballam
Y
Y
Barnes
C
C
D000293:Adolescent; D000328:Adult; D000528:Alternaria / Q000032:analysis*; D002241:Carbohydrates / Q000302:isolation & purification*; D002648:Child; D002845:Chromatography; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005656:Fungal Proteins / Q000302:isolation & purification*; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D008297:Male; D010636:Phenols; D012685:Sepharose; D012882:Skin Tests
D002241:Carbohydrates; D005656:Fungal Proteins; D010636:Phenols; D007073:Immunoglobulin E; D012685:Sepharose