Effect of urea on pI's of ampholytes and casein in isoelectric focusing

✍ Scribed by R.V. Josephson; S.K. Maheswaran; C.V. Morr; R. Jenness; R.K. Lindorfer

• Publisher: Elsevier Science
• Year: 1971
• Tongue: English
• Weight: 436 KB
• Volume: 40
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(71)90408-8

✦ Synopsis

Urea has been used to dissociate and solubilize proteins for isoelectric focusing (electrofocusing) in acrylamide gels (llJ8) and sucrose gradient columns (2,3,4,17). In using concentrated urea (7 M) in electrofocusing experiments with whole bovine casein, we observed that the pH of the ampholyte gradient was shifted upward, thus introducing uncertainty into the apparent isoelectric points (~1's) of the separated proteins. In this communication we report the important role of urea in altering the pl values of the low molecular weight polyamino-polycarboxylic ampholytes and focused proteins.

Methods. Isoelectric focusing experiments were carried out at 10°C in a special glass column (LKB 8101) of 110 ml capacity according to the procedure outlined in the LKB instruction manual (9). A linear sucrose gradient solution of 0 to 47% (w/v), formed by a gradient mixer (LKB 8121), was prepared to contain final concentrations of 1% carrier ampholytes (LKB, pH 3-6), with or without 7 Al urea and 50-80 mg of casein. The light and dense electrode solutions used were dilute NaOH (cathode) and dilute H,S04 (anode), respectively, with or without 7M urea. It has been suggested that cyanate, a frequent contaminant of urea, undergoes carbamylation with proteins and/or ampholytes (12,15). Therefore, Ultra Pure urea (Mann Research Laboratories), free from detectable cyanate, was used in all experiments. Analytical Reagent grade sucrose (Mallinckrodt Chemical Co.) was used to form the density gradients. Class-distilled deionized water was used in all solutions. Focusing was established and maintained at 52.0 W of power as recommended (9) by subjecting the column to an electrode potential of 450V for 24 hr and progressively increasing the potential to 700V to 1 Scientific Journal Series Paper No. 7306, Minnesota Agricultural Experiment Station.