Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing.
We have demonstrated that N-terminal sequencing can be performed successfully 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 affected 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 observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Mo/ller, 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 Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H+K+-ATPase, the sequencing yield after urea treatment increased almost threefold.
Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA et CNRS URA 2096, CEA de Saclay, 91191 Gif sur Yvette Cedex, France.
S
S
Soulié
L
L
Denoroy
J P
JP
Le Caer
N
N
Hamasaki
J D
JD
Groves
M
M
le Maire
D000595:Amino Acid Sequence; D003461:Crystallography; D008565:Membrane Proteins / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D017421:Sequence Analysis / Q000379:methods*; D014508:Urea / Q000737:chemistry / Q000494:pharmacology*
D008565:Membrane Proteins; D009211:Myoglobin; D014508:Urea