Selective extraction, solubilization, and reversed-phase high-performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures.
The number of solvents capable of dissolving myelin and proteolipid protein (PLP) and of being used as a mobile phase for the separation of myelin proteins by reversed-phase high-performance liquid chromatography (RP-HPLC) is very limited. In a thorough study, we found that aqueous tetrahydrofuran (THF) fulfilled such a requirement. The maximal amount of protein extracted corresponded to a THF/water ratio of 4:1 v/v and a polarity index of 5.16. This mixture dissolved a purified PLP preparation completely, 60% of proteins from fresh myelin, and 20% of white matter total proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of those extracts, followed by densitometric analysis, showed that the amount and type of proteins dissolved depended on the polarity (i.e., the content of water) of the solvent mixture used. This selective effect was greater for basic protein in myelin preparations. Crude extracts highly enriched in basic protein can be prepared. In addition, the solvent system THF/water proved to be very useful as a mobile phase in RP-HPLC for separating myelin proteins. Using a C3 column and a linear gradient from 30% to 100% THF in water, both containing 0.1% trifluoroacetic acid (TFA), we separated completely the three main protein fractions of central nervous system (CNS) myelin in a short period of time. The high solubility power of THF/water mixtures prolonged greatly the life of the column.
Díaz
R S
RS
Cajal Institute, C.S.I.C., Madrid, Spain.
Regueiro
P
P
Monreal
J
J
Tandler
C J
CJ
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005663:Furans; D009186:Myelin Sheath / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D012997:Solvents; D014867:Water
D005663:Furans; D009419:Nerve Tissue Proteins; D012997:Solvents; D014867:Water; C018674:tetrahydrofuran