Internal Standards for Molecular Weight Determinations of Proteins by Polyacrylamide Gel Electrophoresis: APPLICATIONS TO ENVELOPE PROTEINS OF ESCHERICHIA COLI

Abstract

Abstract A new technique was devised to be used with molecular weight determinations of proteins on polyacrylamide gels. Internal standards consisting of proteins covalently linked to 1-dimethylaminonaphthalene-5-sulfonyl- (DANS-) groups were prepared. DANS- derivatives of insulin and monomer, dimer, trimer, tetramer, pentamer, and hexamer of egg white lysozyme and serum albumin covered molecular weights from 6,600 to 360,000. They were concurrently applied in small amounts on a single gel together with the unknown sample. Proteins were separated by electrophoresis in 0.5% sodium dodecyl sulfate. Positions of the DANS- proteins were detected under an ultraviolet lamp as yellowish fluorescent bands. These bands are reproducible and related to the positions obtained by standard gel staining techniques. Even 0.2 µg of a DANS-protein was detectable. Molecular weights of unknowns were determined by interpolation from their positions. This method was applied to envelope proteins of Escherichia coli. The molecular weights of major proteins were determined. The molecular weights of Proteins X and Y, which have been shown in a previous paper to be related to cell division and DNA synthesis, respectively, were reexamined and found to be 50,000 and 44,000, respectively. One of the major envelope proteins was found at the molecular weight of 7500. This protein showed a very high arginine to histidine ratio (about 11 times as high as the others) distinctly different from all other proteins of higher molecular weights. Thus, this protein is not a subunit of proteins of higher molecular weights.