Separation of membrane-embedded tryptic peptides of Na,K-ATPase by size-exclusion chromatography.
Several attempts to separate hydrophobic tryptic and cyanogen bromide-digested short peptides from Na,K-ATPase, using HPLC and different acid-organic solvent gradients, failed because of the insolubility of the peptides in the initial or final solvents of the gradients used for elution. Therefore, we opted to use a detergent-containing mobile phase. For sodium dodecyl sulphate-solubilized tryptic peptides of M(r) 7 x 10(3)-100 x 10(3), elution on a TSK-G3000SW size-exclusion column successfully separates families of peptides with a resolution of M(r) 5 x 10(3)-10 x 10(3). Peptides in these size ranges can then be resolved completely by tricine-sodium dodecyl sulphate gel electrophoresis, and identified by microsequencing after transfer to polyvinylidene difluoride paper. For separation of smaller peptides a Biosep-SEC-S2000 column, eluted at slow flow-rates, was evaluated. Use of ammonium chloride buffer allows sensitive detection at 214 nm. The separated fractions are resolved and identified on 16.5% tricine gels. Reasonable resolution has been obtained with defined cyanogen bromide fragments of myoglobin. Resolution of small tryptic and cyanogen bromide fragments of Na,K-ATPase is less successful, but the experiments suggest ways of improving the resolution of peptides in the range M(r) 2 x 10(3)-10 x 10(3).
Tal
D M
DM
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Goldshleger
R
R
Karlish
S J
SJ
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D006868:Hydrolysis; D008567:Membranes, Artificial; D010455:Peptides / Q000302:isolation & purification*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D008567:Membranes, Artificial; D010455:Peptides; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D003488:Cyanogen Bromide