TREATMENT WITH CRYSTALLINE ULTRA-PURE UREA REDUCES THE AGGREGATION OFINTEGRAL MEMBRANE-PROTEINS WITHOUT INHIBITING N-TERMINAL SEQUENCING

We have demonstrated that N-terminal sequencing can be performed succe ssfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not af fected by the presence of urea provided ''ultra-pure'' urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observati ons are useful for the study of integral membrane proteins, in particu lar to study their topology from proteolysis experiments, since heatin g in the presence of urea before SDS-PAGE reduces membrane protein agg regation [Soulie, S,, Moller, J.V., Falson, P., and le Maire, M. (1996 ) Anal, Biochem, 236, 363-364], We show that the sequencing yield of a hydrophobic peptide from reticulum Ca2+-ATPase was more than doubled in the presence of urea in accord with the quantification of the Cooma ssie Blue staining of the gel and of the amount present on the polyvin ylidene difluoride membrane. For three peptides of the gastric H+K+-AT Pase, the sequencing yield after urea treatment increased almost three fold.