Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA.
The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.
S
S
Elsayed
A S
AS
Hammer
M B
MB
Kalvenes
E
E
Florvaag
J
J
Apold
H
H
Vik
D000485:Allergens; D000818:Animals; D000918:Antibody Specificity; D003488:Cyanogen Bromide; D000939:Epitopes; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007122:Immunoelectrophoresis; D007073:Immunoglobulin E / Q000276:immunology; D007526:Isoelectric Point; D008970:Molecular Weight; D010047:Ovalbumin / Q000276:immunology*; D010434:Pepsin A; D010446:Peptide Fragments / Q000276:immunology; D011489:Protein Denaturation; D011852:Radioallergosorbent Test; D014357:Trypsin
D000485:Allergens; D000939:Epitopes; D010446:Peptide Fragments; D007073:Immunoglobulin E; D010047:Ovalbumin; D014357:Trypsin; D010434:Pepsin A; D003488:Cyanogen Bromide