SEQUENCE-ANALYSIS OF WHEAT-GRAIN ALLERGENS SEPARATED BY 2-DIMENSIONALELECTROPHORESIS WITH IMMOBILIZED PH GRADIENTS

Micropreparative two-dimensional (2-D) gel electrophoresis with immobi lized pH gradients (4-8) in the first dimension (IPG-DALT) was optimiz ed for the separation of salt-soluble wheat grain proteins associated with bakers' asthma disease. The resolved polypeptides were electroblo tted onto a polyvinylidene difluoride (PVDF) membrane and incubated wi th the pooled sera from four asthmatic bakers. Bound IgE was demonstra ted by alkaline phosphatase conjugated anti-human IgE. Major IgE bindi ng was detected in the 27 kDa, 37 kDa and, to a lesser extent, in the 14-18 kDa area of the 2-D immunoblots, respectively. Since the main pu rpose of our study was to determine the N-terminal amino acid sequence s of the major wheat grain allergens, N-terminal sequencing was perfor med for six out of a total of eleven major allergens located in the 27 kDa area, for one out of two 37 kDa allergens, and for two out of fou r 14-18 kDa allergens. Our results revealed that two of the 27 kDa pol ypeptides are clearly related to several Acyl-CoA oxidase variants of barley and rice, whereas no significant homologies were found for the remaining four 27 kDa allergens analyzed. The N-terminus of the 37 kDa allergen appeared to be blocked so that no sequence information was o btained, while the two 14-18 kDa allergens analyzed were identified as members of the wheat a-amylase-inhibitor family.