Additives for immobilized pH gradient two-dimensional separation of particulate material: comparison between commercial and new synthetic detergents.
We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commercial compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3-12, the concentration of urea compatible with 2% detergent increases from 4.5 M for the parent molecule up to 7 M. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl-N,N-dimethylammonio-3-propanesulfonate of a major protein (pI = ca. 5.5, Mr = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a pK of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)
School of Pharmaceutical Sciences, Milano, Italy.
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E
Gianazza
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T
Rabilloud
L
L
Quaglia
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P
Caccia
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Astrua-Testori
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L
Osio
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Grazioli
P G
PG
Righetti
D000818:Animals; D002417:Cattle; D003902:Detergents; D004910:Erythrocyte Membrane / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000032:analysis; D007525:Isoelectric Focusing / Q000379:methods*; D007668:Kidney / Q000032:analysis; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification*; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods; D010944:Plants / Q000032:analysis; D051381:Rats
D003902:Detergents; D008565:Membrane Proteins