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 aci d-organic solvent gradients, failed because of the insolubility of the peptides in the initial or final solvents of the gradients used for e lution. Therefore, we opted to use a detergent-containing mobile phase . For sodium dodecyl sulphate-solubilized tryptic peptides of M(r) 7 . 10(3)-100 . 10(3), elution on a TSK-G3000SW size-exclusion column suc cessfully separates families of peptides with a resolution of M(r) 5 . 10(3)-10 . 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 difluo ride paper. For separation of smaller peptides a Biosep-SEC-S2000 colu mn, 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 resoluti on has been obtained with defined cyanogen bromide fragments of myoglo bin. Resolution of small tryptic and cyanogen bromide fragments of Na, K-ATPase is less successful, but the experiments suggest ways of impro ving the resolution of peptides in the range M(r) 2 . 10(3)-10 . 10(3) .