METALLOPROTEIN ANALYSIS BY CAPILLARY ISOELECTRIC FOCUSING

Authors: Richards, Mark; HUANG, TUNG-LIANG - BECKMAN INSTRUMENTS, INC

Submitted to: The Journal of Chromatography B: Biomedical Applications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/1996
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 objective of this study was to apply the technique of capillary isoelectric focusing (cIEF) to the separation of three metalloproteins: transferrin (a plasma iron- binding protein), conalbumin (the egg form of transferrin) and metallothionein (a heavy metal binding protein). A fast, simple and reliable technique was developed for the separation of different forms (isoforms) of these three proteins. The information derived from this study is useful in characterizing changes in the molecular structure of a wide variety of metalloproteins based on the absence or presence of bound metal ions. In addition, information concerning the nature of transferrin isoforms has importance to clinical diagnoses of diseases affecting iron metabolism.

Technical Abstract: Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3-10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe3+/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3-10 gave a similar separation pattern to conalbumin with four major peaks at pIs of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (diferic forms). Rabbit liver MT subjected to cIEF with a pH gradient of 3-10 gave a complex separation pattern with two prominent peaks (pIs of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.