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Title: DEVELOPMENT OF A HIGH PERFORMANCE CAPILLARY ISOELECTRIC FOCUSING TECHNIQUE WITH APPLICATION TO STUDIES OF MICROHETEROGENEITY IN CHICKEN CONALBUMIN

Author
item TUNG-LIANG, HUANG - BECKMAN INSTRUMENTS
item Richards, Mark

Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/2/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Capillary electrophoresis is a newly emerging technique that is beginning to be applied to the analysis of complex biomolecules such as proteins, peptides and nucleic acids. It involves the separation of molecules in narrow bore capillaries with very high voltage applied that causes them to move in the electric field according to their net charge. Recently, a new mode of capillary electrophoresis has been developed which employs isoelectric focusing (IEF). This technique (IEF) separates proteins and peptides based on subtle differences in their net charge caused by alteration in their amino acid content/sequence or by the presence of a substance such as a metal ion or carbohydrate bound to the protein or peptide. The objectives of this study were: 1) to develop and optimize the technique of capillary isoelectric focusing (CIEF) and 2) to apply IEF to the analysis of a specific iron-binding protein that occurs naturally in egg white. A fast, simple and reliable technique was developed and CIEF has now been commercialized in kit form by Beckman Instruments Inc. In addition, we were able to determine the iron binding properties of conalbumin based on differential migration of the iron-bound forms of this protein. Thus, not only was a new technique successfully developed, but it was also shown to be applicable to a specific type of analysis useful in characterizing egg composition.

Technical Abstract: A robust, simple, reproducible isoelectric focusing method using capillary electrophoresis that exhibits high stability and migration time reproducibility, and pH linearity over a wide pH gradient was developed. Consecutive runs (over 113 runs) of proteins and peptide with pIs ranging from 9.45 to 2.75 yielded excellent yielded excellent migration time reproducibility (< 2% RSD). 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 microheterogeneties in chicken conalbumin (ovotransferrin), an iron-binding protein in egg white. Conalbumin (low iron) separated into three major components with pIs of 7.2, 6.6, and 6.2. When the protein was saturated with iron (2 Fe/mol), a shift to lower pIs was detected. Chicken serum transferrin subjected to CIEF gave a pattern similar to iron-free conalbumin with three pIs 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.