Unfolding of human serum transferrin in urea studied by high-performance capillary electrophoresis.
High-performance capillary electrophoresis (HPCE) was used to monitor the progress of the unfolding of human serum transferrin in urea. Denaturation curves of the transferrin forms were constructed plotting the migration times corrected for the viscosity vs. the concentration of urea in the buffer. The practical advantage of capillary zone electrophoresis is the short analysis time, 5-15 min, as compared with slab-gel experiments, which require overnight runs for similar purposes. The resolution increased with the urea concentration, and hence high concentrations are beneficial for quantitative and qualitative analysis of mixtures of transferrin forms. Unfolding intermediates of the isoforms, which interconvert to the unfolded state slowly compared with the time scale of the electrophoretic separation, and also the completely unfolded isoforms were resolved and detected simultaneously when iron-free transferrin was subjected to denaturation by urea at concentrations between 3 and 6 M. However, no unfolding intermediates were observed with transferrin isoforms containing two iron atoms (i.e. diferric transferrin molecules), which accordingly are strongly resistant to urea denaturation. The unfolding of the transferrin isoforms depends on the iron content of the complexes, but not the carbohydrate content. HPCE in the presence of urea in this mode has the potential to become an analytical tool for diagnosis of diseases in which the transferrin patterns change.
Kilár
F
F
Institute of Biochemistry, Uppsala University, Sweden.
Hjertén
S
S
D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D006801:Humans; D007501:Iron / Q000737:chemistry; D007536:Isomerism; D011489:Protein Denaturation; D017510:Protein Folding; D014168:Transferrin / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
D014168:Transferrin; D014508:Urea; D007501:Iron