Development of a high-performance capillary isoelectric focusing technique with application to studies of microheterogeneity in chicken conalbumin.
A robust, simple, reproducible isoelectric focusing method using capillary electrophoresis that exhibits high stability, migration time reproducibility and pH linearity over a wide pH gradient was developed. Consecutive runs (over 113 runs) of several proteins and one peptide with isoelectric points (p/s) ranging from 9.45 to 2.75 yielded excellent migration time reproducibility (< 2% R.S.D.). Experimental parameters including buffer aging and capillary-to-capillary variation were thoroughly examined and optimized to improve the migration time reproducibility. The capillary isoelectric focusing (CIEF) method was applied to the analysis of chicken conalbumin (ovotransferrin), an iron-binding protein in egg white. Conalbumin (low iron content) separated into three major components with p/s of 7.2, 6.6 and 6.2. When the protein was saturated with iron (2 Fe/mol), a shift to lower p/s was detected. Chicken serum transferrin subjected to CIEF gave a pattern similar to conalbumin with three p/s of 7.1, 6.6 and 6.1, indicating that it was not fully saturated with iron. Thus, CIEF can be used as a potential analytical method to provide information about the metal-binding properties of specific metalloproteins.
Huang
T L
TL
Beckman Instruments Inc., Fullerton, CA 92634, USA.
Richards
M
M
D000818:Animals; D002021:Buffers; D002645:Chickens; D003207:Conalbumin / Q000032:analysis* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007525:Isoelectric Focusing / Q000379:methods*; D016014:Linear Models; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000032:analysis
D002021:Buffers; D014168:Transferrin; D003207:Conalbumin; D007501:Iron