EFFECT OF SEPARATION CONDITIONS ON AUTOMATED ISOELECTRIC-FOCUSING OF CARBOHYDRATE-DEFICIENT TRANSFERRIN AND OTHER HUMAN ISOTRANSFERRINS USING THE PHASTSYSTEM

To investigate the effect of automated isoelectric focusing conditions in the PhastSystem, e.g., the point of sample application, prerun and separation times, and minimized gels on isotransferrin band pattern, human sera were analyzed with native transferrin iron load, after iron saturation or iron depletion in vitro. Varying the focusing condition s we found (i) Point of sample application (anode, middle of the gel, cathode) strongly affected transferrin iron loss. It was greatest at t he anode and least at the cathode. (ii) Without pre run, distinct tran sferrin iron loss also occurred, A short prerun time prevented iron lo ss, but increasing it did not improve transferrin iron load stability as stated by others. (iii) An inappropriately long separation time ine vitably yielded iron loss. In conclusion, inappropriate isoelectric fo cusing conditions strongly affect iron load stability of isotransferri ns (obviously via low pH within the gel), resulting in transferrin iro n release and cofocusing of isotransferrins with different sialic acid or iron contents. For determination of carbohydrate-deficient transfe rrin, such conditions resulted in overestimation of the marker of chro nic alcohol abuse. Our findings may be of guiding importance for isoel ectric focusing of protein-ligand complexes. We recommend the procedur e described for development of isoelectric focusing of protein-ligand complexes. (C) 1995 Academic Press, Inc.